Spinosyn formulations for treatment of Demodex-induced ocular and facial conditions

ABSTRACT

The present invention concerns materials and methods for treating an ocular  Demodex  mite infestation of an eye of a human or animal subject, or for treating a condition of the eye or skin associated with ocular  Demodex  mite infestation, the method comprising topically administering a composition comprising one or more spinosyn compounds to the ocular surface (conjunctiva and/or cornea) or other anatomical structure of the eye, or to an area adjacent the eye.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/523,984, filed Jul. 26, 2019, which claims the benefit of U.S.Provisional Application Ser. No. 62/711,309, filed Jul. 27, 2018, whichis hereby incorporated by reference herein in its entirety, includingany FIGURES, tables, nucleic acid sequences, amino acid sequences, ordrawings.

BACKGROUND OF THE INVENTION

Demodex is a genus of ecto-parasitic mites within the Arachnida classthat live in or near hair follicles of mammals. Demodex mites have acommensal relationship with humans, and they asymptomatically parasitizethe pilosebaceous follicles of humans. Thus, these mites are part of thenormal microbiome in the sebaceous areas of the skin in humans (Grice EA et al., “The skin microbiome: potential for novel diagnostic andtherapeutic approaches to cutaneous disease,” Semin Cutan Med Surg.,2014, 33(2):98-103). In healthy skin, the density of the mites isnormally low, and this low density does not cause skin diseases. Whenthe density of these mites increases, they induce facial skinconditions. Patients with an ocular Demodex infestation often complainof itching, burning, redness, crusting at the base of the lashes, blurryvision and dry eye.

Among the over 100 species of Demodex mites that have been identified,Demodex folliculorum and Demodex brevis are the two main speciescommonly found on humans (Lacey N et al., “Under the lash: Demodex mitesin human diseases,” Biochem (Lond)., 2009 Aug. 1, 31(4):2-6). Mostindividuals become colonized during childhood and numbers proliferate inand around the pilosebaceous units at the age of puberty as levels ofsecreted sebum increase, and increasing in prevalence with age (Elston DM, “Demodex mites: facts and controversies”, Clin Dermatol, 2010,28(5):502-504). This may be due to lipases produced by Demodex, allowingthe mite to utilize sebum as a food source (Jimenez-Acosta F et al.,“Demodex mites contain immunoreactive lipase”, Arch Dermatol, 1989,125(10):1436-1437). Other nutritional sources include cellular debrisand bacteria that reside in and around the pilosebaceous unit.

D. folliculorum has a comparatively long body and proliferates at thebase of the lashes (cylindrical dandruff), causing anterior blepharitis.Specifically, D. folliculorum consume epithelial cells at the hairfollicle causing lash distention, hypoplasia, and reactivehyperkeratinization, which is commonly observed as trichiasis andmadarosis. D. brevis is shorter in size and burrows into the sebaceousand meibomian glands, causing posterior blepharitis. D. brevis obstructsmeibomian gland openings, leading to insufficient tear lipid secretion,which can be an inflammatory trigger for dry eye.

Demodex mites also carry their own bacterial reservoirs that cancontribute to ocular surface inflammation. The bacteria on the surfaceof the mite has been shown to compete with known staphylococcal species,producing a human host immune response leading to an inflammatory eyelidand periorbital epidermal to subepidermal reaction. If the disease is ina chronic and progressive phase, the inflammation may spread to theconjunctiva and cornea, potentially leading to infiltrativekeratoconjunctivitis, nodular scar deposition and cornealneovascularization.

BRIEF SUMMARY OF THE INVENTION

The present invention concerns materials and methods for treating anocular Demodex mite infestation of an eye of a human or animal subject,or for treating a condition of the eye or skin associated with ocularDemodex mite infestation, such as Demodex folliculorum, Demodex brevis,or both.

One aspect of the invention concerns a method for treating an ocularDemodex mite infestation of an eye of a human or animal subject, or fortreating a condition of the eye or skin associated with ocular Demodexmite infestation, comprising topically administering a compositioncomprising one or more spinosyn compounds to the ocular surface(conjunctiva and/or cornea) or other anatomical structure of the eye, orto an area adjacent the eye. In some embodiments, the condition is oneor more from among Demodex-induced blepharitis (also called Demodexblepharitis), Demodex-induced ocular rosacea, Demodex-induced facialrosacea, dry eye, meibomian gland dysfunction, chalazion, hordeolum,follicular inflammation, non-specific facial dermatitis, infiltrativekeratoconjunctivitis, nodular scar deposition, or cornealneovascularization. The treatment can alleviate one or more signs orsymptoms in the subject selected from among: itching, burning, foreignbody sensation, crusting and redness of the lid margin, blurry vision,cylindrical dandruff, eyelash misalignment, eyelash trichiasis, eyelashmadarosis, lid margin inflammation, meibomian gland dysfunction,blepharoconjunctivitis, and blepharokeratitis in the subject.

In some embodiments, the composition is topically administered to anarea adjacent to the eye, wherein the area comprises one or more of aneyelid, eyelid margin, eyelashes, eyelash follicles, eyebrow, or eyebrowfollicles. In some embodiments, the composition is topicallyadministered to an area adjacent to the eye, wherein the area comprisesa sebaceous gland opening of the eyelid (e.g., one or more of gland ofZeis, gland of Moll, or Meibomian gland).

Another aspect of the invention concerns a topical compositioncomprising 0.1% to 10% (w/v) of the one or more spinosyn compounds (alsoreferred to herein as the “topical composition” or “spinosyncomposition”, or “topical spinosyn composition” of the invention), andmethods for making the topical composition. In some embodiments, thecomposition is a solution, suspension, salve, spray, lotion, gel, paste,balm, foam, mousse, scrub or cleanser (e.g., shampoo or soap), cream, orointment. Soaps may be solid, such as a bar, liquid, or semi-solid.

Another aspect of the invention concerns an article of manufacturecomprising the topical composition; and a container with the topicalcomposition contained therein. In some embodiments, the container is acollapsible or non-collapsible tube, bag, packet, blister, strip,ampoule, viral, bottle, can, or jar.

Another aspect of the invention concerns an ocular or facial applicatorpre-treated with, or containing, the topical composition. In someembodiments, the applicator is a swab, cosmetic pad, wipe, wipe stick,towelette, sponge, gauze, puff, wand, brush, or comb.

Another aspect of the invention concerns a kit useful for treating anocular Demodex mite infestation of an eye of a human or animal subject,or for treating a condition of the eye or skin associated with ocularDemodex mite infestation, such as Demodex folliculorum, Demodex brevis,or both. The kit comprises the topical composition; and an ocular orfacial applicator. Optionally, the applicator may be pretreated with, orcontain the topical composition. The kit may further include a containercontaining the composition. The kit may further include instructions(e.g., printed or digital instructions) for treating an ocular Demodexmite infestation of an eye of a human or animal subject, or for treatinga condition of the eye or skin associated with ocular Demodex miteinfestation, by topically administering the composition to the ocularsurface (conjunctiva and/or cornea) or other anatomical structure of theeye, or to an area adjacent the eye.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure (Formula (I)) of some embodiments ofspinosyn compounds that may be used in the compositions and methods ofthe inventions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns materials and methods for treating anocular Demodex mite infestation of an eye of a human or animal subject,or for treating a condition of the eye or skin associated with ocularDemodex mite infestation, such as Demodex folliculorum, Demodex brevis,or both.

One aspect of the invention concerns a topical composition useful fortreating an ocular Demodex mite infestation of an eye of a human oranimal subject, or for treating a condition of the eye or skinassociated with ocular Demodex mite infestation, the compositioncomprising 0.1% to 10% (w/v) of the one or more spinosyn compounds.

The fermentation product identified in U.S. Pat. No. 5,362,634 as A83543is a family of related compounds produced by Saccharopolyspora spinosa.These compounds have been referred to as factors or components A, B, C,D, E, F, G, H, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, Y, and the like(also see published international patent application WO 93/09126 and WO94/20518) and are referred to as spinosyn A, B, C, and so on. Spinosynsare a family of macrocyclic lactones having pesticidal activity on avariety of pests. The early identified spinosyns were found to have a5,6,5-tricylic ring system, fused to a 12-membered macrocyclic lactone,a neutral sugar (rhamnose), and an amino sugar (forosamine) (see Kirst,H. A. et al. (1991) “A83543A-D, Unique Fermentation-Derived TetracyclicMacrolides” Tetrahedron Letters 32(37):4839-4842). Natural spinosyns maybe produced via fermentation from cultures deposited as NRRL 18719,18537, 18538, 18539, 18743, 18395, and 18823 of the stock culturecollection of the Midwest Area Northern Regional Research Center,Agricultural Research Service, United States Department of Agriculture,1815 North University Street, Peoria, Ill. 61604. Spinosyns are alsodisclosed in U.S. Pat. Nos. 5,496,931, 5,670,364, 5,591,606, 5,571,901,5,202,242, 5,767,253, 5,840,861, 5,670,486 and 5,631,155. Spinosyns havebeen found useful for the control of arachnids, nematodes, and insects.

Examples of spinosyns and spinosoids (semi-synthetic analogs) aredisclosed in Kirst H A, “The spinosyn family of insecticides: realizingthe potential of natural products research,” The Journal of Antibiotics,2010, 63:101-111; Legocki J et al., “Contemporary trends in developmentof active substances possessing the pesticidal properties: spinosyninsecticides,” Pestycydy/Pesticides, 2010, 1-4:59-71; Sparks T C et al.,“Natural products as insecticides: the biology, biochemistry andquantitative structure-activity relationships of spinosyns andspinosoids”, Pest Manag Sci, 2001, 57:896-905; and Salgado V L et al.,“Studies on the Mode of Action of Spinosad: The Internal EffectiveConcentration and the Concentration Dependence of Neural Excitation,”Pesticide Biochemistry and Physiology,” 1998, 60:103-110, which are eachincorporated herein by reference in their entirety).

Spinosyn compounds useful in the various aspects and embodiments of theinvention refer to spinosyns and spinosoids having miticidal activityagainst members of the Demodex genus. The terms “acaricidal” and“miticidal” are used herein interchangeably to refer to the ability tokill mites of the Demodex genus in any life stage, or the ability tointerfere with a Demodex mite's growth or life cycle in any way thatresults in an overall reduction in Demodex mite population. In someembodiments, the Demodex species is D. folliculorum, D. brevis, or both.For example, the term “miticidal” includes inhibition or elimination ofreproductive ability of a pest, as well as inhibition of a pest fromprogressing from one form to a more mature form, e.g., transitionbetween various larval forms or transition from larvae to proto-nymph orfrom proto-nymph to nymph, or from nymph to adult. Further, the termmiticidal is intended to include all phases of a mite life cycle; thus,for example, the term includes larvicidal, ovicidal, and adulticidalaction (see, for example, Rather P. A. et al., Human Demodex Mite: TheVersatile Mite of Dermatological Importance”, Indian J Dermatol, 2014,January-February, 59(1):60-66). Techniques for assessing in vitro and invivo Demodex killing activity are known in the art and may be utilized(see, for example, U.S. Pat. No. 8,128,968 (Gao et al.); Gao Y-Y et al.,“In vitro and in vivo killing of ocular Demodex by tea tree oil,” Br JOphthalmol, 2005, 89:1468-1473; and Tighe S et al., “Terpinen-4-ol isthe most active ingredient of tea tree oil to kill Demodex mites,” TransVis Sci Tech., 2013, 2(7):2).

In some embodiments, the one or more spinosyn compounds have thechemical structure of Formula (I), shown in FIG. 1, or apharmaceutically acceptable salt thereof, where R¹, R², R³, R⁴, R⁵, R⁶,R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, can be independentlyselected from the group consisting of: null; H; F; Cl; Br; I; OH; CN;(C₁₋₄)alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl; (C₂₋₄)alkenyl, such as ethenyl,propenyl, butenyl, where the double bond can be located at any positionin the alkenyl carbon chain, and including any alkenyl conformationalisomers; alkynyl; aralkyl; alkaryl; halogenated alkyl; heteroalkyl;aryl; heterocyclyl; cycloalkyl; cycloalkenyl; cycloalkynyl;hydroxyalkyl; aminoalkyl; amino; alkylamino; arylamino; dialkylamino;alkylarylamino; diarylamino; acylamino; hydroxyl; thiol; thioalkyl;alkoxy; alkylthio; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro;carbamoyl; trifluoromethyl; phenoxy; benzyloxy; phosphonic acid;phosphate ester; sulfonic acid (—SO₃H); sulfonate ester; sulfonamide;alkaryl; arylalkyl; carbamate; amino; alkylamino; arylamino;dialkylamino; alkylarylamino; diarylamino; alkylthio; heteroalkyl;alkyltriphenylphosphonium; heterocyclyl; ketone (═O); ether (—OR¹⁷); andester (—COOR¹⁸ and —OC(═O)R¹⁸);

where R⁵ and R⁷ can be a double bond within the cyclopentane ring;

where R¹¹ and R¹³ can be a double bond within the cyclohexane ring;

where R¹⁷ can be independently selected from the group consisting of: a(C₁₋₄)alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl; (C₂₋₄)alkenyl, such as ethenyl,propenyl, butenyl, where the double bond can be located at any positionin the alkenyl carbon chain, and including any alkenyl conformationalisomers; and alkynyl;

where R¹⁸ can be independently selected from the group consisting of: a(C₁₋₄)alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl; (C₂₋₄)alkenyl, such as ethenyl,propenyl, butenyl, where the double bond can be located at any positionin the alkenyl carbon chain, and including any alkenyl conformationalisomers; and alkynyl.

In some embodiments, the composition comprises spinosyn A, spinosyn D,or a combination thereof. The chemical structures of these and manyother spinosyn compounds have been known for some time. Spinosyn A hasthe following substituent atoms or groups in the structure of Formula(I): R¹, R²═(C₁₋₄)alkyl, namely methyl; R³=ketone (═O); R⁴=null; R⁵ andR⁷ are a double bond within the cyclopentane ring; R⁸, R⁹, R¹⁰═—OR¹⁷,namely —OCH₃; R¹¹ and R¹³ are a double bond within the cyclohexane ring;R¹² and R¹⁴ are H; R¹⁵=null; and R¹⁶=ketone (═O). Spinosyn D has thefollowing substituent atoms or groups in the structure of Formula (I):R²═(C₁₋₄)alkyl, namely methyl; R³=ketone (═O); R⁴=null; R⁵ and R⁷ are adouble bond within the cyclopentane ring; R⁸, R⁹, R¹⁰═—OR¹⁷, namely—OCH₃; R¹¹ and R¹³ are a double bond within the cyclohexane ring; R¹² is(C₁₋₄)alkyl, namely methyl; R¹⁴ is H; R¹⁵=null; and R¹⁶=ketone (═O).

In some embodiments, the spinosyn A and spinosyn D are present in thecomposition in a ratio of about 18:2 to about 16:4 spinosyn A tospinosyn D. In some embodiments, the spinosyn A and spinosyn D arepresent in the composition in a ratio of about 17:3 spinosyn A tospinosyn D (spinosad).

The composition may be formulated in any dosage form that is useful fortopical administration to the ocular surface (conjunctiva and/orcornea), other anatomical structure of the eye, and/or to an areaadjacent the eye, such as an eyelid, eyelid margin, eyelashes, eyelashfollicles, eyebrow, or eyebrow follicles, or an area encompassing asebaceous gland opening of the eyelid (e.g., one or more of gland ofZeis, gland of Moll, or Meibomian gland). In some embodiments, thecomposition is a solution, suspension, salve, spray, lotion, gel, paste,balm, foam, mousse, scrub or cleanser (e.g., shampoo or soap), cream, orointment. A soap may be solid, such as a bar, liquid, or semi-solid.

Methods for making dermatologically acceptable and/or ophthalmicallyacceptable compositions may be employed to mix the one or more spinosyncompounds with one or more additional components of the composition. Thecomponents of the composition may be pre-sterilized, or the compositionmay be sterilized during or at the end of production.

In some embodiments, the composition may be formed as a cosmetic product(e.g., make-up), such as foundation, mascara, eye shadow, or eye liner.The composition may also be in the form of a mask or make-up remover.

In various embodiments, the compositions encompassed herein comprisepharmaceutically acceptable excipients such as those listed inRemington: the Science and Practice of Pharmacy 866-885 (Alfonso R.Gennaro ed. 19th ed. 1995; Ghosh, T. K.; et al. Transdermal And TopicalDrug Delivery Systems, which is hereby incorporated by reference hereinin its entirety), including, but not limited to, protectives,adsorbents, demulcents, emollients, preservatives, antioxidants,moisturizers, buffering agents, solubilizing agents, skin-penetrationagents, and surfactants.

Preferably, the spinosyn composition includes a dermatologicallyacceptable and/or ophthalmically acceptable base. As used herein, a“dermatologically acceptable base” refers to one or more non-detergentexcipients that do not cause irritation, inflammation, pain, or otherharm to the skin when applied to the skin adjacent the eye at effectiveconcentrations. As used herein, an “ophthalmically acceptable base”refers to one or more non-detergent excipients that do not irritate orotherwise harm the surface of the eye when topically administered toocular surface at effective concentrations.

Protectives and adsorbents include, but are not limited to, dustingpowders, zinc stearate, collodion, dimethicone, silicones, zinccarbonate, aloe vera gel and other aloe products, vitamin E oil,allantoin, glycerin, petrolatum, and zinc oxide. Demulcents include, butare not limited to, benzoin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, and polyvinyl alcohol. Emollients include, but are notlimited to, animal and vegetable fats and oils, myristyl alcohol,

alum, and aluminum acetate. Preservatives include, but are not limitedto, chlorine dioxide, quaternary ammonium compounds, such asbenzalkonium chloride, benzethonium chloride, cetrimide, dequaliniumchloride, and cetylpyridinium chloride; mercurial agents, such asphenylmercuric nitrate, phenylmercuric acetate, and thimerosal;alcoholic agents, for example, chlorobutanol, phenylethyl alcohol, andbenzyl alcohol; antibacterial esters, for example, esters ofparahydroxybenzoic acid; and other antimicrobial agents such aschlorhexidine, chlorocresol, benzoic acid and polymyxin. Suitableantioxidants include, but are not limited to, ascorbic acid and itsesters, sodium bisulfate, butylated hydroxytoluene, butylatedhydroxyanisole, tocopherols, and chelating agents like EDTA and citricacid. Suitable moisturizers include, but are not limited to, glycerin,sorbitol, polyethylene glycols, urea, and propylene glycol. Suitablesolubilizing agents include, but are not limited to, quaternary ammoniumchlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates.Suitable skin-penetration agents include, but are not limited to, ethylalcohol, isopropyl alcohol, octylphenylpolyethylene glycol, oleic acid,polyethylene glycol 400, propylene glycol, N-decylmethylsulfoxide, fattyacid esters (e.g., isopropyl myristate, methyl laurate, glycerolmonooleate, and propylene glycol monooleate); and N-methylpyrrolidone.

The composition may include one or more excipients. In some embodiments,the composition includes one or more excipients selected from amongpetrolatum, mineral oil, propylparaben, methylparaben, lanolin,chlorobutanol, water, lanolin alcohol, sodium thiosulfate, sodiumphosphate monobasic, phenylmercuric acetate, mannitol, zinc chloride,sodium phosphate, potassium acetate, hypromelloses, gentamcicin sulfate,boric acid, sodium hydroxide, lanolin oil, carbomer homopolymer type b(allyl pentaerythritol crosslinked), or benzalkonium chloride. In someembodiments, the composition includes no parabens.

The composition may include a gelling agent. Examples of gelling agentsinclude gum, agar, carrageenan, petrolatum, or a cellulosic polymer(e.g., hydroxyethyl cellulose or hydroxymethyl cellulose).

The composition may further include one or more of a solvent,co-solvent, demulcent, emollient, preservative, antimicrobial agent,antioxidant, moisturizer, stabilizing agent, or solubilizing agent.

In some embodiments, the composition includes one or more excipientsselected from among water, sodium hydroxide, polysorbate 80, glycerine,castor oil, sodium acetate, boric acid, sorbic acid, edetate disodium,carbomer copolymer type A or B (allyl pentaerythritol crosslinked), orsilicone oil or silicone polymer gel (e.g., dimethicone orcyclomethicone).

Mucoadhesive polymers that provide localized delivery of the activeingredient to the anatomical target site may be included, and areespecially useful in gels. Such polymers have a property known asbioadhesion, which refers to the capacity for a drug carrier to aspecific biological tissue, such as a mucous membrane. These polymerscan extend the contact time of the active agent with the biologicaltissue and thereby improve bioavailability. Examples of bioadhesivepolymers having various mucoadhesive performance qualities includecarboxymethylcellulose, carbopol, polycarbophil, and sodium alginate.

The pH of the spinosyn composition should be in a dermatologicallyand/or ophthalmically acceptable range. Normal tears have a pH of about7.4 and possess some buffer capacity. The administration of acomposition to the eye stimulates the flow of tears and the rapidneutralization of any excess hydrogen or hydroxyl ions within the buffercapacity of the tears. Where only 1 or 2 drops of a liquid compositioncontaining an active agent are added to the eye, the buffering action ofthe tears is usually adequate to raise the pH and prevent markeddiscomfort.

While it is desirable for an ophthalmic composition to have the same pHand and isotonic value as lacrimal fluid, this is not usually possible.The buffer system should be selected that is nearest to thephysiological pH of 7.4 and does not compromise miticidal efficacy ofthe composition. Verification of the pH of the mixture and adjustmentwith a solution of a buffer or neutralizing agent, and also theincorporation of the optional additives, may be carried out, accordingto their chemical nature, during one of the steps of the method ofpreparation. In some embodiments, the spinosyn composition includes oneor more dermatologically and/or ophthalmically acceptable pH adjustingagents or buffering agents, including, but not limited to, acids such asacetic, boric, citric, lactic, phosphoric and hydrochloric acids; basessuch as sodium hydroxide, sodium phosphate, sodium borate, sodiumcitrate, sodium acetate, sodium, lactate andtris-hydroxymethylaminomethane; and buffers such as citrate/dextrose,sodium bicarbonate and ammonium chloride. Such acids, bases and buffersare included in an amount required to maintain pH of the composition ina dermatologically and/or ophthalmically acceptable range.

In some embodiments, the composition comprises no additional agent thathas activity against the Demodex mite. In some embodiments, thecomposition includes no active agent other than the one or more spinosyncompounds.

In some embodiments, where possible, the composition may includepharmaceutically acceptable salts of compounds in the composition, suchas spinosyn compounds and other compounds when present. In someembodiments, the composition comprises acid addition salts of thecompound(s). In some embodiments, the composition comprises baseaddition salts of the compounds(s). As used herein, the termpharmaceutically acceptable salts or complexes refers to salts orcomplexes (e.g., solvates, polymorphs) that retain the desiredbiological activity of the parent compound and exhibit minimal, if any,undesired toxicological effects.

In some embodiments, the composition further comprises an additionalagent having miticidal activity against the Demodex mite, such as one ormore agents selected from among ivermectin, pyrethrin, pyrethroid (e.g.,permethrin, resmethrin, or D-phenothrin), tea tree oil (TTO), TTOcomponent (e.g., terpinen-4-ol (T4O)), metronidazole, hypochlorous acid(HOCl), essential oil (e.g., peppermint oil or Salvia), alkali metalsalt (e.g., lithium salt), phosphorothioate (e.g., a non-volatile, fatsoluble phosphorothioate such as coumaphos), or formamidine (e.g.,amitraz).

In some embodiments, the composition further comprises one or moreantibiotic agents. In some embodiments, the antibiotic agent hasantibacterial activity against one or more bacteria found on or withinthe Demodex mite, such as Streptococci, Staphylococci, or Bacillusoleronius. In some embodiments, the antibiotic agent also has miticidalactivity, such as metronidazole.

In some embodiments, the composition further comprises one or moreanti-inflammatory agents. Examples of anti-inflammatory agents that maybe included are glucocorticoid or other steroid (e.g., prednisone,cortisone acetate, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone acetate,deoxycorticosterone acetate, aldosterone), non-steroidalanti-inflammatory drug (e.g., salicylates, arylalkanoic acids,2-arylpropionic acids, N-arylanthranilic acids, oxicams, coxibs, orsulphonanilides), Cox-2-specific inhibitor (e.g., valdecoxib, celecoxib,or rofecoxib), leflunomide, gold thioglucose, gold thiomalate, aurofin,sulfasalazine, hydroxychloroquinine, minocycline, TNF-alpha bindingprotein (e.g., infliximab, etanercept, or adalimumab), abatacept,anakinra, interferon-beta, interferon-gamma, interleukin-2, allergyvaccine, antihistamine, antileukotriene, beta-agonists, theophylline, oranticholinergic, antibiotics, tacrolimus, or retinoid.

In some embodiments, the anti-inflammatory agent is formulated fortopical use. In some embodiments, the topical anti-inflammatory agent isa topical steroid. In the U.S., topical steroids are classified(Class/Group I-VII) by their ability to constrict capillaries and causeskin blanching, with Group I being the strongest or most potent, andGroup VII being the weakest and mildest. Some examples of topicallyformulated steroids that may be utilized include clobetasol,betamethasone, diflorasone, fluocinonide, flurandrenolide, halobetasole,amcinonide, desoximetasone, halcinonide, fluticasone, triamcinolone,fluocinolone, hydrocortisone, mometasone, triamcinolone, alclometasone,denoside, and prednicarbate.

In some embodiments, the anti-inflammatory agent is formulated forophthalmic use. Some examples of ophthalmically formulated steroids thatmay be utilized include dexamethasone, difluprednate, fluoromethalone,loteprednol etabonate, and rimexolone.

Optionally, the composition may include an abrasive agent foradministration to areas of skin and hair adjacent to the eye. Inclusionof the abrasive may serve to mechanically agitate an area of tissueadjacent the eye, eyelid, eyelash, and follicle to improve access of theone or more spinosyn compounds in the composition to the Demodex. Thetreatment may work to agitate or move unwanted material such as Demodexeggs, larva or mites and remove them from the eyelash follicle or otherpart of the eye or eyelid, or to make them accessible to the spinosyncomposition. The abrasive may also eliminate organisms by direct killingor damage. The abrasive may include any abrasive particle, powder, orcrystal including but not limited to one or more of the following:aluminum oxide (e.g., alumina, aluminum trioxide, corundum powder),barium sulfate, boron nitride, calcium carbonate, cellulose acetate,ceramic, diamond, diatomaceous earth, emerald, ethylene/acric acidcopolymer, fibers, garnet, glass, kaolin, lauroyl lysine, lava,magnesium oxide, mica, modified starch, nylon, other metals, otherpolymers, other silicon dioxides or silicon containing materials,polyethylene, polymethyl methacrylate polypropylene, polystyrene,polytetrafluoroethylene (PTFE), pumice, ruby, sand, sapphire, seashells,sericite, silica, silicon dioxide, silicon carbide, sodium bicarbonate,sodium chloride crystals, starch, silk, talc, topaz, zeolite, or polymerparticles.

An abrasive particle may be any shape and have any number of sides. Anabrasive particle may be overall diamond (triangle) shaped, elliptical,marquise shaped, octagonal, oval, pear shaped, rectangular, round,squared, or may be combinations or variations (e.g., a rounded square)of these shapes. An abrasive particle may have one surface or may havemore than one surface (e.g., sides or faces). An abrasive particle mayhave 2, 3, 4, 5, 6-10, 11 to 20, 21 to 30, up to 40, up to 50, up to 60,or more than 60 sides. A surface of an abrasive particle may besubstantially smooth, regular, textured or irregular. An abrasiveparticle may have one or more sharp edges or points. An abrasiveparticle may be sized from about 1 to about 600 microns across a longestdimension.

Abrasive particles in a group may all be similarly shaped to one anotheror may be differently shaped from one another. Abrasive particles in agroup may all be about the same size, or may range in size. A group ofparticles may be larger than a minimum or may be smaller than a maximum.A group of abrasive particle may include particles from about 1 to about15 microns across, about 15 microns to about 25 microns, about 25microns to about 100 microns, about 100 to about 300 microns, or about300 to about 600 microns. In one example, a group of abrasive particlesmay include particles from greater than about 25 microns to less thanabout 300 microns across. Differently sized and differently shapedparticles may be chosen for different reasons. Different sizes andshapes may be chosen for different eye area conditions, different skintypes or sensitivities, and/or different methods of application. Aparticle with a rough surface may be applied using an applicator, suchas a wand or towelette, while a substantially round particle may bepropelled towards the surface under pressure.

An abrasive may include a group of separate particles, or may include asubstrate with an abrasive surface or may be any combination orvariation. A substrate may have a plurality of abrasive particlesconnected with (attached to) it to provide an abrasive surface, or itmay be a material having a rough textured surface. A rough texturedsurface may have a pore size on its surface from about 1 to about 15microns across, about 15 microns to about 25 microns, about 25 micronsto about 100 microns, about 100 to about 300 microns, or about 300 toabout 600 microns.

Ointments

In some embodiments, a dermatologically and/or ophthalmically acceptablebase includes a pharmaceutically acceptable ointment base. Examples ofsuitable ointment bases include, but are not limited to, oleaginousointment bases such as petrolatum (e.g., liquid petrolatum or whitepetrolatum), plastibase, hard paraffin, white soft paraffin, yellow softparaffin, liquid paraffin, emulsifying wax, microcrystalline wax, whitebees wax, yellow bees wax, carnauba wax, wool wax (wool fat), mineraloil, olive oil, purified lanolin, anhydrous lanolin, and water solubleointment bases such as polyethylene glycol (e.g., polyethylene glycol400 or polyethylene glycol 3350), propylene glycol, polyoxyethylene,polyoxypropylene, or any combinations thereof.

In some embodiments, the composition is an ointment and includes one ormore excipients selected from among petrolatum, mineral oil,propylparaben, methylparaben, lanolin, chlorobutanol, water, lanolinalcohol, sodium thiosulfate, sodium phosphate monobasic, phenylmercuricacetate, mannitol, zinc chloride, sodium phosphate, potassium acetate,hypromelloses, gentamcicin sulfate, boric acid, sodium hydroxide,lanolin oil, carbomer homopolymer type B (allyl pentaerythritolcrosslinked), or benzalkonium chloride.

In some embodiments, the spinosyn composition is an ointment having apetrolatum base. Petrolatum is a semi-solid mixture of hydrocarbons thatprovides certain advantages for skin and ophthalmic applications.Petrolatum is a pseudoplastic, which provides solid state behavior,which increases stability and prevents phase separation and settling ofsuspended particles. Petrolatum is thixotropic, which is excellent forretaining a drug in suspension while retaining spreadability. Forophthalmic uses, this permits spreading in the eye and as shear force isapplied (when blinking), the product becomes more fluid and retainsfluidity, helping to coat the eye. Because petrolatum is non-aqueous, itspreads and softens but will not be flushed out of the eye with tears(unlike aqueous solutions). As an non-aqueous and non-hygroscopicexcipient, issues with hydrolysis and hydrolytic degradation ofcomponents can be avoided. As an insoluble excipient, petrolatum limitsthe treatment to local delivery, with little if any system absorption.Petrolatums for pharmaceutical products are classified as Petrolatum USPand White Petrolatum USP. For ophthalmic uses, while petrolatums aretypically preferred. Petrolatums vary in color and clarity, as well asrheological properties (consistency, flow, and yield stress). Additionalexcipients such as mineral oil, surfactants, and preservatives can lowerthe apparent viscosity and yield stress relative to their concentrationin the formulation.

Emulsions

The compositions of the invention may be formulated as an emulsion,e.g., an oil-in-water emulsion. The topical oil-in-water emulsioncompositions may be in liquid, paste, or solid form, or in the form ofointments, creams, gels, sprays, foams, suspensions, lotions, shampoos,or washing bases. The compositions may also be in the form ofsuspensions of microspheres or nanospheres or of lipid or polymericvesicles or of polymeric patches and of hydrogels for controlledrelease. These compositions for topical application may be in anhydrousform, in aqueous form or in the form of an emulsion.

In some embodiments of the invention, the composition is in the form ofan emulsion of the cream or lotion type, of a gel, or of a solution, andmore particularly in the form of a cream.

In some embodiments, the emulsion includes one or more excipientsselected from among water, sodium hydroxide, polysorbate 80, glycerine,castor oil, carbomer copolymer type A, sodium acetate, boric acid,sorbic acid, edetate disodium, or silicone oil or silicone polymer gel(e.g., dimethicone or cyclomethicone).

Conventional emulsions are typically relatively unstable, virtuallyhomogeneous systems of two immiscible liquids, one of which is dispersedin the other in the form of fine droplets (micelles). This dispersion isstabilized by virtue of the action of surfactant-emulsifiers whichmodify the structure and the ratio of the forces at the interface, andtherefore increase the stability of the dispersion by decreasing theinterface tension energy. Surfactant emulsifiers are amphiphiliccompounds possessing a hydrophobic component having affinity for oil anda hydrophilic component having affinity for water, thus creating a linkbetween the two phases. Ionic or nonionic emulsifiers thereforestabilize oil/water emulsions by adsorbing to the interface and forminglamellar layers of liquid crystals.

The compositions of the invention may contain one or more non-ionicsurfactant emulsifiers. The emulsifier power of non-ionic surfactants isclosely linked to the polarity of the molecule. This polarity is definedby the HLB (hydrophilic/lipophilic balance). Conventional emulsions aregenerally stabilized by a mixture of surfactants, the HLBs of which canbe quite different but the proportion of which in the mixturecorresponds to the required HLB of the fatty phase to be emulsified.

Suitable non-ionic surfactant emulsifiers can be selected from the groupconsisting of Cetostearyl alcohol, Cetyl alcohol, Cocamide DEA, CocamideMEA, Isoceteth-20, Oleyl alcohol, Sorbitan monostearate, Sorbitantristearate, Stearyl alcohol, tyloxapol, softigen, solutol HS15,poloxamers such as Pluronic F-68LF™ or Lutrol F68, Pluronic L-62LF™ andPluronic L62D™ (BASF Wyandotte Corp., Parsippany, N.J., USA),polysorbates such as polysorbate 20 and polysorbate 80, polyoxyethylenefatty acid esters such as Emulphor™ (GAF Corp., Wayne, N.J., USA).

In some embodiments, the emulsion includes a carbomer homopolymer(Carbopol), which can emulsify oil-in-water type suspensions, allowingfor non-cloudy/non-sticky formulations.

In some embodiments, the compositions of the invention are oil-in-wateremulsions that comprise: (a) one or more spinosyn compounds; (b) an oilyphase comprising a fatty substance and/or an emollient; (c) water; and,optionally, (d) a surfactant emulsifier.

Examples of fatty substances include vegetable, mineral, animal orsynthetic oils, silicone oils, Guerbet alcohols or other substances, andmixtures thereof.

Examples of mineral oils include paraffin oils of various viscosities,such as Primol 352, Marcol 82 or Marcol 152 marketed by Esso. Examplesof vegetable oil include sweet almond oil, palm oil, soybean oil, sesameoil and sunflower oil. Examples of animal oils include lanolin oil,squalene oil, fish oil, and mink oil. Examples of synthetic oils includeesters, such as cetearyl isononanoate marketed in particular under thename Cetiol SN by Cognis France, diisopropyl adipate, for instance, theproduct marketed under the name Ceraphyl 230 by ISF, isopropylpalmitate, for instance the product marketed under the name Crodamol IPPby Croda, or caprylic capric triglyceride such as Miglyol 812 marketedby Huls/Lambert Riviere. Examples of silicone oils include dimethicone,such as the product marketed under the name Dow Corning 200 fluid, andcyclomethicone, such as the product marketed under the name Dow Corning244 fluid by Dow Corning, and the product marketed under the nameMirasil CMS by SACI-CFPA.

Other fatty substances may include fatty acids such as white petrolatum,stearic acid, fatty alcohols such as stearyl alcohol, cetostearylalcohol and cetyl alcohol, or derivatives thereof, waxes such asbeeswax, carnauba wax or candelilla wax, and also gums, in particularsilicone gums.

One or more fatty substances in the composition may be present in anamount of about 20% to about 40% by weight of the composition. In someembodiments, the one or more fatty substances are present in an amountof about 20% to about 35% by weight of the composition.

The oily phase of the composition can include one or more emollients,such as vegetable, mineral, animal or synthetic oils, silicone oils,isopropyl palmitate, 1-decene polymer (hydrogenated), C₁₂-C₁₅ alkylbenzoate, C₁₂-C₁₅ alkyl benzoates esters, lanolin alcohol and isopropylmyristate; and mixtures thereof. Emollients in the composition may bepresent in an amount of about 10% to about 20% by weight of thecomposition.

In some embodiments, the oily phase of the composition comprises amixture of fatty substances and emollients, and in some embodiments, atleast two emollients. In some embodiments, the fatty substances arewhite petrolatum and mineral oil and the emollients are lanolin alcoholand isopropyl myristate.

The oily phase of the emulsion may be present at a content of from 15 to45% by weight relative to the total weight of the composition, andpreferably from 20 to 40% by weight.

The composition may comprise up to 15% by weight of a suitablesurfactant emulsifier. In some embodiments, the surfactant emulsifier ispresent in the composition in from about 5% to about 15% by weight ofthe composition.

The composition may comprise from 0.1% to 10% of one or more spinosynsby weight relative to the total weight of the composition. In someembodiments, the composition comprises from 0.5% to 10% of one or morespinosyns by weight relative to the total weight of the composition.

The composition of the invention may also contain water or bufferranging from 50% to 95%. In some embodiments, the composition containsfrom 55% to 80% water or buffer by weight relative to the total weightof the composition. The water used in the composition according to theinvention is preferably purified or distilled water.

The composition may also contain inert additives or combinations ofadditives, such as flavor enhancers; preservatives; stabilizers;humidity regulators; pH regulators; osmotic pressure modifiers; UV-A andUV-B screening agents; and antioxidants. In some embodiments, theoil-in-water emulsion composition of spinosyn is devoid of preservingagents.

These additives may be present in the composition at a concentrationfrom 0.001% to 20% by weight relative to the total weight of thecomposition.

In some embodiments, the compositions are oil-in-water emulsions thatcomprise: (a) one or more spinosyns in an amount of about 0.01% to about10% by weight; (b) an oily phase comprising one or more fatty substancesin an amount of about 20% to about 40% by weight and one or moreemollients in an amount of about 10% to about 20% by weight; (c) one ormore surfactant emulsifiers in an amount of about 5% to about 15% byweight; and (d) water.

Exemplified Formulations of the Composition

In some embodiments, the composition is a cream, wherein in addition tothe one or more spinosyn compounds (e.g., spinosad), the compositionincludes one or more of the following ingredients: water or buffer,mineral oil, acetylated lanolin alcohol, stearyl alcohol, cetearylalcohol, and propylene glycol. In some embodiments, the cream includeseach of the foregoing ingredients.

In some embodiments, the composition is a cream, wherein in addition tothe one or more spinosyn compounds (e.g., spinosad), the compositionincludes one or more of the following ingredients: water or buffer,propylene glycol, mineral oil, actylated lanolin alcohol, cetyl alcohol,stearyl alcohol, cetearyl alcohol, and polyoxyl 35 castor oil. In someembodiments, the cream includes each of the foregoing ingredients.

In some embodiments, the composition is a lotion, wherein in addition tothe one or more spinosyn compounds (e.g., spinosad), the compositionincludes one or more of the following ingredients: water or buffer,isopropyl myristate, castor oil, polyoxyl 40 stearate, cetearyl alcohol,and cetyl esters wax. In some embodiments, the lotion includes each ofthe foregoing ingredients.

In some embodiments, the composition is an ointment, wherein in additionto the one or more spinosyn compounds (e.g., spinosad), the compositionincludes one or more of the following ingredients: water or buffer,propylene glycol, castor oil, acetylated lanolin alcohol, cetyl alcohol,stearyl alcohol, cetearyl alcohol, cetyl esters wax, povidone K 90, andstearic acid. In some embodiments, the ointment includes each of theforegoing ingredients.

In some embodiments, the composition is an ointment, wherein in additionto the one or more spinosyn compounds (e.g., spinosad), the compositionincludes one or more of the following ingredients: propylene glycol,mineral oil, acetylated lanolin alcohol, stearyl alcohol, glycerylstearate/PEG-100 stearate, polyoxyl 40 stearate, and stearic acid. Insome embodiments, the ointment includes each of the foregoingingredients.

In some embodiments, the composition is one described by one of Tables1-5. In some embodiments, for a given composition, the sum of each %concentration of the listed ingredients in the table is 100%.

In some embodiments, the composition is one described by one of Tables1-5, but with one or more of the listed excipients substituted for oneor more different excipients having the same listed function(s).

In some embodiments, in each of Tables 1-5, using 0.1% spinosad as areference point, for each incremental w:w % decrease of spinosadconcentration, the same incremental w:w % increase is made to theingredient that is dominant in the formulation (i.e., the ingredienthaving the greatest minimum %). Conversely, for each incremental w:w %increase of spinosad, there is a decrease by the same w:w % from theexcipient that is dominant in the formulation. As an example, in Table1, for each 0.1% increase in spinosad from 0.1% spinosad, there would bea corresponding decrease of 0.1% in distilled water or buffer. As afurther example, in Table 5, for each 1.0% increase in spinosad from0.1%, there would be a corresponding decrease of 1% in mineral oil.

TABLE 1 Creams Active/Excipient Function Range (w:v %) Spinosad Active0.1%-10% Distilled Water or buffer Solvent, pH adjuster, vehicle    54-90 part of the cream base Mineral oil Emollient, moisterizer,    15-97 solvent Acetylated lanolin alcohol Solubilizer and emulsifier     5-8 Cetyl esters wax Thickener and emollient     10-10.3 Stearylalcohol emulsion stabilizer,      5-15 surfactant/emulsifying agent,viscosity increasing agent. Cetearyl alcohol Emulsifier and thickener     7-8 Propylene glycol Solvent      3-71

TABLE 2 Creams Active/Excipient Function Range (w:v %) Spinosad Active0.1-10% Distilled Water or buffer Solvent, pH adjuster,  35-90 vehiclepart of the cream base Propylene glycol solvent  15-71 Mineral oilEmollient, moisterizer,  14-97 solvent Acetylated lanolin alcoholSolubilizer and emulsifier   6-8 Cetyl alcohol Emulsifier, thickener  8-12 Stearyl Alcohol emulsion stabilizer,  11-15surfactant/emulsifying agent, viscosity increasing agent. Cetearylalcohol Emulsifier, solubilizer,   7-8 thickener Polyoxyl 35 castor oilSurfactant, emulsifying   4-4.86 agent, solubilizing agent

TABLE 3 Lotions Active/Excipient Function Range (w:v %) Spinosad Active0.1-10% Distilled Water or buffer Solvent, pH adjuster,  70-90 vehiclepart of the cream base Isopropyl myristate solvent   3-15 Castor oilSolvent, vehicle, skin  10-15 softener Polyoxyl 40 stearate Solubilizerand emulsifier   2-8.8 Cetearyl alcohol Emulsifier, solubilizer,   4-8thickener Cetyl esters wax Thickener and emollient  10-10.3

TABLE 4 Ointments Active/Excipient Function Range (w:v %) SpinosadActive 0.1-10% Distilled Water or buffer Solvent, pH adjuster,  13-90vehicle part of the cream base Propylene glycol solvent  30-71 Castoroil Emollient, moisterizer,  15-97 solvent Acetylated lanolin alcoholSolubilizer and emulsifier   5-8 Cetyl alcohol Emulsifier, thickener 10-12 Stearyl Alcohol emulsion stabilizer,  10-15surfactant/emulsifying agent, viscosity increasing agent. Cetearylalcohol Emulsifier, solubilizer,   5-8 thickener Cetyl esters waxThickener and emollient   5-10.3 Povidone K 90 Thickener, gelling   1-2polymer Stearic acid Surfactant, emulsifying   5-22.5 agent, thickener

TABLE 5 Ointments Active/Excipient Function Range (w:v %) SpinosadActive 0.1-10% Propylene glycol solvent  32-71 Mineral oil Emollient,moisturizer,  44-97 solvent Acetylated lanolin alcohol Solubilizer andemulsifier   5-8 Straryi Alcohol emulsion stabilizer,   8-15surfactant/emulsifying agent, viscosity increasing agent. Glycerylstearate/PEG-100 Emulsifier, solubilizer   3-7.5 stearate Polyoxyl 40stearate Solubilizer and   2-8.8 emulsifying agent Stearic acidSurfactant, emulsifying   5-22.5 agent, thickenerMethods of Treatment

An aspect of the invention is a method for treating an ocular Demodexmite infestation of an eye of a human or animal subject, or for treatinga condition of the eye or skin associated with ocular Demodex miteinfestation, comprising topically administering a composition comprisingone or more spinosyn compounds to the ocular surface (conjunctiva and/orcornea) or other anatomical structure of the eye, or to an area adjacentthe eye.

Without being limited by theory as to potential mechanism of action, foran existing Demodex infestation, the goal of treatment as a therapy istypically to reduce the number of Demodex mites in an anatomical locussuch as the ocular surface (conjunctiva and/or cornea) or otheranatomical structure of the eye, or to an area adjacent the eye, so thatany one or more signs of a Demodex-associated condition will bealleviated or eliminated. As Demodex mites represent a part of thenormal skin microbiome, and the level of sensitivity to the Demodexmites and immunoreactive factors associated with the mites, may varybetween subjects, the goal of therapy may be to reduce the number ofliving Demodex mites to a level that is normal or is healthy for thatindividual (below a threshold of parasitic over-population), or toeradicate all Demodex mites from the anatomical locus or loci targeted.

The composition may also be topically administered prophylactically to asubject without a Demodex infestation in order to prevent or delay onsetof a Demodex infestation.

Through one or more administrations, an amount of a spinosyn compoundthat is sufficient to reduce the number of Demodex mites in a anatomicallocus, as compared to a corresponding anatomical locus in the absence ofthe amount or concentration of the spinosyn or other miticide, isdelivered (a miticidally effective amount).

The composition is to be topically administered to the ocular surface(conjunctiva and/or cornea) or other anatomical structure of the eye, orto an area adjacent the eye, which may be done by the subject (i.e.,self-administered) or by another individual.

In some embodiments, for formulations intended for administration to theocular surface, depending on its viscosity, the composition may beadministered dropwise (e.g., as with an eye dropper), or placed at thecorner of the eye or eyelid, or onto the inside of the upper or lowereyelid. For example, one or more drops (of, for example, about 30microliters each) may be administered. The subject may blink tofacilitate distribution of the composition on the ocular surface. Insome embodiments, the composition is topically administered to theocular surface,

In some embodiments, the composition is topically administered to theocular surface, or other anatomical structure of the eye, or to an areaadjacent the eye, immediately before going to sleep.

In some embodiments, the composition is topically administered to anarea adjacent to the eye, wherein the area comprises a sebaceous glandopening of the eyelid (e.g., one or more of gland of Zeis, gland ofMoll, or Meibomian gland).

In some embodiments, the composition is topically administered to one ormore structures of epidermal invaginations known as pilosebaceous units(e.g., hair, hair follicle, sebaceous gland).

The composition may be topically administered using an ocular or facialapplicator, such as those described herein, or using a part of the body,such as a finger or knuckle (by wiping, rubbing, massaging, etc.).

Examples of applicators include a swab, cosmetic pad, wipe, wipe stick,towelette, sponge, gauze, puff, wand, brush, or comb. In someembodiments, the applicator includes at least a portion that makescontact with the target anatomical area and may be composed of woven ornon-woven materials such as cotton, polyester, or rayon. Applicators maybe disposable or reusable.

The spinosyn composition may be administered topically by gentleapplication to one or more anatomical sites on a subject, including thesubject's ocular surface or other anatomical structure of the eye, or toan area adjacent to the eye such as an eyelid, eyelid margin, eyelashes,eyelash follicles, eyebrow, or eyebrow follicles, or to a sebaceousgland opening of the eyelid (e.g., one or more of gland of Zeis, glandof Moll, or Meibomian gland). The spinosyn composition may be massagedonto the skin. In some embodiments, the spinosyn composition is left onthe area until the next treatment. In other embodiments, excess spinosyncomposition is wiped away or washed away after administration.

Optionally, one or more anti-inflammatory agents may be administered tothe subject before, during, or after administration of the spinosyncomposition, which can assist in suppressing or reducing inflammationthat may be caused by some embodiments of the spinosyn composition or byany mechanical irritation that may be applied as part of treatment. Ananti-inflammatory agent may also suppress or reduce potentialinflammation associated with the decaying mites. Anti-inflammatoryagents may be administered to the subject by a route appropriate to theformulation. For example, an anti-inflammatory agent may be administeredtopically, included in the spinosyn composition or topicallyadministered in a separate composition. In other embodiments, ananti-inflammatory agent may be administered to the subject systemically(e.g., orally).

Examples of anti-inflammatory agents that may be administered include aglucocorticoid or other steroid (e.g., prednisone, cortisone acetate,prednisolone, methylprednisolone, dexamethasone, betamethasone,triamcinolone, beclometasone, fludrocortisone acetate,deoxycorticosterone acetate, aldosterone), non-steroidalanti-inflammatory drug (e.g., salicylates, arylalkanoic acids,2-arylpropionic acids, N-arylanthranilic acids, oxicams, coxibs, orsulphonanilides), Cox-2-specific inhibitor (e.g., valdecoxib, celecoxib,or rofecoxib), leflunomide, gold thioglucose, gold thiomalate, aurofin,sulfasalazine, hydroxychloroquinine, minocycline, TNF-alpha bindingprotein (e.g., infliximab, etanercept, or adalimumab), abatacept,anakinra, interferon-beta, interferon-gamma, interleukin-2, allergyvaccine, antihistamine, antileukotriene, beta-agonists, theophylline, oranticholinergic, antibiotics, tacrolimus, or retinoid.

Optionally, the anatomical site may be scrubbed prior to treatment withthe spinosyn composition, as a pre-treatment or preparatory step, withan agent such as a cleansing substance. An abrasive may be applied toareas adjacent to the eye to remove skin, a layer of skin, skin debris,micro-organisms, eyelashes, oils, and/or other related or unrelatedsubstances. The abrasive may mechanically agitate area of tissueadjacent the eye, eyelid, eyelash, and follicle to improve access of thespinosyn composition to the Demodex. The treatment may work to agitateor move unwanted material such as Demodex eggs, larva or mites andremove them from the eyelash follicle or other part of the eye oreyelid, or to make them accessible to the spinosyn composition. Thedermabrasive may also eliminate organisms by direct killing or damage.The abrasive may include any abrasive particle, powder, or crystalincluding but not limited to one or more of the following: aluminumoxide (e.g., alumina, aluminum trioxide, corundum powder), bariumsulfate, boron nitride, calcium carbonate, cellulose acetate, ceramic,diamond, diatomaceous earth, emerald, ethylene/acric acid copolymer,fibers, garnet, glass, kaolin, lauroyl lysine, lava, magnesium oxide,mica, modified starch, nylon, other metals, other polymers, othersilicon dioxides or silicon containing materials, polyethylene,polymethyl methacrylate polypropylene, polystyrene,polytetrafluoroethylene (PTFE), pumice, ruby, sand, sapphire, seashells,sericite, silica, silicon dioxide, silicon carbide, sodium bicarbonate,sodium chloride crystals, starch, silk, talc, topaz, zeolite, or polymerparticles.

Optionally, before, during, or after administration of the spinosyncomposition, an additional agent with activity against Demodex may beadministered. In some embodiments, the agent has activity againstDemodex, such as ivermectin, pyrethrin, pyrethroid (e.g., permethrin,resmethrin, or D-phenothrin), tea tree oil (TTO), TTO component (e.g.,terpinen-4-ol (T4O)), metronidazole, hypochlorous acid (HOCl), essentialoil (e.g., peppermint oil or Salvia), alkali metal salt (e.g., lithiumsalt), phosphorothioate (e.g., a non-volatile, fat solublephosphorothioate such as coumaphos, also identified as O,O-DiethylO-3-chloro-4-methyl-2-oxo-2H-chromen-7-yl phosphorothioate), orformamidine (e.g., amitraz, also identified asN,N′-[(Methylimino)dimethylidyne]di-2,4-xylidine). The additional agentmay be administered as a component of the spinosyn composition, oradministered in a separate composition, topically or by another route.

The dosage regimen to treat, prevent, or ameliorate the condition(s) forwhich relief is sought, can be modified in accordance with a variety offactors. These factors include the disorder from which the subjectsuffers, as well as the age, weight, sex, diet, and medical condition ofthe subject. Thus, the dosage regimen actually employed can vary widelyand therefore can deviate from the dosage regimens set forth herein.

Detection of Demodex

The methods of the invention may further comprise confirming thepresence of Demodex mites on the subject prior to administering thespinosyn composition to the subject. Several clinical signs can alertone to the presence of Demodex in the lashes. Appreciation of theseclues can lead one to perform one of two simple examination techniquesthat will confirm the presence of the mites.

1. Cylindrical dandruff (CD). This is the most obvious clinical sign andan excellent indicator for mite infestation. Cylindrical dandruff isvisible at the slit lamp, and it is different than the scaly, scurf-likedebris seen with traditional anterior blepharitis.

2. Alterations of the skin surrounding the lash follicle. The skin nearthe opening of the follicle becomes distended and raised, possibly witha greasy, oily appearance to the skin surrounding the follicle.

3. Changes in lash appearance. In longstanding infestations, the lashesbecome thin and brittle, or they may begin to lose their color.Misdirection and loss of lashes may also occur.

4. Lid hyperemia/telangiectasia. Because the presence of mites isassociated with increases in interleukins and different types ofpro-inflammatory cytokines, increases in levels of lid inflammation maybe apparent, which may lead to increased vascularization along the lidmargin.

5. Patient history/associations with other disease. Prevalence ofDemodex increases with age, and a strong association in patients withacne rosacea, seborrheic dermatitis, and other forms of inflammatoryskin conditions has been observed, including allergic symptoms such asitching.

These clinical signs can alert to the presence of Demodexoverpopulation. However, each of these items is not diagnostic in itsown right—with the possible exception of cylindrical dandruff.Confirmation of the presence of mites, as well as a method ofcategorizing the degree of mite infestation can be done through one oftwo simple steps: lash epilation (e.g., two to four different lashesfrom the lid along with associated debris/cylindrical dandruff, viewingthe complex under a light microscope) and lash rotation (transferringdebris from the follicle to a slide and examining under a lightmicroscope or visualizing under high magnification at a slit lamp(25-40×).

Accordingly, the presence of Demodex mites may be confirmed by one ormore techniques, such as direct mite identification and measurement ofmite density, e.g., presence of cylindrical dandruff at eyelash root,skin alteration surrounding lash follicle, changes in lashes, and lashsampling (ash epilation with light microscope or lash rotation with slitlamp). In addition to ex vivo depilation and microscopy, in vivoconfocal microscopy may be used (Randon M et al., “In vivo confocalmicroscopy as a novel and reliable tool for the diagnosis of Demodexeyelid infestation,” Br J Ophthalmol, 2015, 99(3):336-341, which isincorporated herein by reference in its entirety).

Molecular diagnostic techniques may be used to detect Demodex-associatedmolecules at the genetic level and/or protein level in biologicalsamples taken from the subject from areas where the mites can be found(e.g., eyelashes, skin surface biopsies). For example, polymerase chainreaction (PCR) or immunoassay (e.g., enzyme-linked immunosorbent assay(ELISA)) may be used to detect and quantify target Demodex nucleic acidor protein molecules in skin and/or hair samples from the subject, suchas exoskeletal chitin, chitin synthase, 62-kDa and 83-kDa antigen ofBacillus oleronius (see, for example, Zhao Ya-e et al., “Cloning andsequence analysis of chitin synthase gene fragments of Demodex mites,” JZheijang Univ Sci B, 2012, 13(10):763-768; Zhao Ya-e et al.,“Discrimination between Demodex folliculorum (Acari: Demodicidae)isolates from China and Spain based on mitochondrial cox/sequences,” JZheijang Univ Sci B, 2013, 14(9):829-836; Hu L et al., “Molecularidentification of four phenotypes of human Demodex in China,” ExpParisitol, 2014, 142:38-42; Li J et al., “Correlation between ocularDemodex infestation and serum immunoreactivity to Bacillus proteins inpatients with facial rosacea,” Ophthalmology, 2010, 117(5):870-877;Szkaradkiewicz A et al., “Bacillus oleronius and Demodex miteinfestation in patients with chronic blepharitis,” Clinical Microbiologyand Infection, 2012, 18(10):1020-1025; Kasetsuwan N et al., “Prevalenceof ocular demodicosis among patients at Tertiary Care Center, Bangkok,Thailand,” Int J Ophthalmol, 2017, 10(1):122-127; Liu D et al., “Demodex(Hair Follicle Mite)”, Chapter 72 in Molecular Detection of HumanParasitic Pathogens, CRC Press, 2012, pages 741-750, which are eachincorporated herein by reference in their entirety).

Thus, in some embodiments of the methods of the invention, confirmationof the presence of Demodex mites comprises identifying the Demodex mitesin vivo or in a biological sample (e.g., a hair or skin sample) ex vivo,or detecting a molecule associated with Demodex mites (e.g., chitin,chitin synthase, lipase, Demodex-associated bacterial antigen (e.g.,Bacillus antigen such as B. oleronius antigen), Demodex 16S rDNA,Demodex 18S rDNA). For example, Demodex mites have been shown to produceimmune-reactive lipase, which may be used as a target for detection ofthe mite (Jimenez-Acosta F. et al., “Demodex mites contain immunoreactive lipase,” Arch Dermatol., 1989, 125:1436-7). Target DNA and RNAmay be extracted and amplified and used qualitatively as molecularbarcodes for identification and inter-species and intra-speciesdifferentiation, and used quantitatively to assess extent ofinfestation. Gene or protein microarray-based analysis may also beutilized. Biological samples from the subject such as skin surface,hair, tears, and peripheral blood may be analyzed for the presence andlevels of inflammatory markers that are consistent with Demodexinfestation.

To monitor the progress of a treatment or to confirm its efficacy, thepresence or absence of Demodex mites or a threshold amount of Demodexmites on a subject may be carried out on a subject-derived sample aftera course of treatment for a period of time to determine whether theDemodex have been reduced to normal levels or been eradicated by thetreatment.

The detection step may be used for any purpose for which detection ofDemodex (e.g., D. folliculorum and/or D. brevis) is desirable, includingdiagnostic and prognostic applications, such as in laboratory andclinical settings. Appropriate samples include any biological samples inwhich Demodex may be found, including clinical samples obtained from amedical subject (human) or veterinary subject (non-human animal).

When the target molecule associated with Demodex mites within a sampleis one that may be targeted by antibodies, such as a protein orcarbohydrate (e.g., chitin, chitin synthase, lipase, Demodex-associatedbacterial antigen (e.g., Bacillus antigen such as B. oleronius antigen),immunochemical techniques using antibodies, either polyclonal ormonoclonal, or antigen-binding fragments of such antibodies, may beused. These immunochemical techniques can involve eitherradioimmunoassay or other well-established assay techniques, suchenzyme-linked immunosorbent assay (ELISA). Target molecules can also bemeasured by standard non-immunochemical techniques such as gaschromatography.

Detecting a target nucleic acid in a sample (e.g., D. folliculorumand/or D. brevis nucleic acid, or Demodex-associated bacterial nucleicacid, or subject immune molecule) using genetic-based moleculardiagnostic techniques typically involves contacting the sample with atleast one probe that is capable of hybridizing to the target nucleicacid, such as a D. folliculorum and/or D. brevis nucleic acid, underconditions of very high stringency, and detecting hybridization betweenthe target nucleic acid and the probe. Detection of hybridizationbetween the probe and the target nucleic acid indicates the presence ofthe target nucleic acid (and thus, the organism, e.g., D. folliculorumand/or D. brevis) in the sample.

In some embodiments, D. folliculorum and/or D. brevis nucleic acidspresent in a sample are amplified prior to using a hybridization probefor detection. For instance, it can be advantageous to amplify a portionof the D. folliculorum and/or D. brevis nucleic acid, and then detectthe presence of the amplified D. folliculorum and/or D. brevis nucleicacid.

Detecting the amplified product typically includes the use of labeledprobes that are sufficiently complementary and hybridize to theamplified target nucleic acid sequence. Thus, the presence, amount,and/or identity of the amplified product can be detected by hybridizinga labeled probe, such as a fluorescently labeled probe, complementary tothe amplified product. In one embodiment, the detection of a targetnucleic acid sequence of interest, such as a D. folliculorum and/or D.brevis nucleic acid includes the combined use of PCR amplification and alabeled probe such that the product is measured using real-time PCR. Inanother embodiment, the detection of an amplified target nucleic acidsequence of interest includes the transfer of the amplified targetnucleic acid to a solid support, such as a blot, for example a Northernblot, and probing the blot with a probe, for example a labeled probe,that is complementary to the amplified target nucleic acid sequence. Inyet another embodiment, the detection of an amplified target nucleicacid sequence of interest includes the hybridization of a labeledamplified target nucleic acid to probes disclosed herein that arearrayed in a predetermined array with an addressable location and thatare complementary to the amplified target nucleic acid.

Any nucleic acid amplification method can be used to detect the presenceof Demodex in a sample. In one specific, non-limiting example,polymerase chain reaction (PCR) is used to amplify the target nucleicacid sequences. In other specific, non-limiting examples, real-time PCR,reverse transcriptase-polymerase chain reaction (RT-PCR), real-timereverse transcriptase-polymerase chain reaction (rt RT-PCR), ligasechain reaction, or transcription-mediated amplification (TMA) is used toamplify the target nucleic acid. In a specific example, the D.folliculorum and/or D. brevis nucleic acid is amplified by real-timePCR, for example real-time TAQMAN® PCR. Techniques for nucleic acidamplification are well-known to those of skill in the art.

Typically, at least two primers are utilized in the amplificationreaction, Amplification of the Demodex nucleic acid involves contactingthe Demodex nucleic acid with one or more primers that are capable ofhybridizing to and directing the amplification of a Demodex nucleic acid(such as a primer capable of hybridizing under very high stringencyconditions to D. folliculorum and/or D. brevis.

The amplified Demodex nucleic acid, can be detected in real-time, forexample by real-time PCR, in order to determine the presence, and/or theamount of Demodex-specific nucleic acid (e.g., D. folliculorum and/or D.brevis) in a biological sample. In this manner, an amplified nucleicacid sequence, such as an amplified D. folliculorum and/or D. brevisnucleic acid sequence, can be detected using a probe specific for theproduct amplified from the Demodex sequence of interest.

A fluorophore is a chemical compound, which when excited by exposure toa particular stimulus such as a defined wavelength of light, emits light(fluoresces), for example at a different wavelength (such as a longerwavelength of light). Fluorophores are part of the larger class ofluminescent compounds. Luminescent compounds include chemiluminescentmolecules, which do not require a particular wavelength of light toluminesce, but rather use a chemical source of energy. Therefore, theuse of chemiluminescent molecules (such as aequorin) eliminates the needfor an external source of electromagnetic radiation, such as a laser.

A probe comprises an isolated nucleic acid capable of hybridizing to atarget nucleic acid (such as Demodex nucleic acid, for example D.folliculorum and/or D. brevis ribosomal nucleic acid molecule). Adetectable label or reporter molecule can be attached to a probe.Typical labels include radioactive isotopes, enzyme substrates,co-factors, ligands, chemiluminescent or fluorescent agents, haptens,and enzymes.

Methods for labeling and guidance in the choice of labels appropriatefor various purposes are discussed, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989) and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley-Intersciences (1987).

One of ordinary skill in the art will know suitable methods forextracting nucleic acids such as RNA and/or DNA from a sample; suchmethods will depend upon, for example, the type of sample in which theDemodex nucleic acid is found. For example, the nucleic acids may beextracted using guanidinium isothiocyanate, such as the single-stepisolation by acid guanidinium isothiocyanate-phenol-chloroformextraction of Chomczynski et al. (Anal. Biochem., 1997, 162:156-59). Thesample can be used directly or can be processed, such as by addingsolvents, preservatives, buffers, or other compounds or substances.Nucleic acids can be extracted using standard methods. For instance,rapid nucleic acid preparation can be performed using a commerciallyavailable kit (such as the QIAGEN® DNA Mini kit (QIAGEN®) Roche MagNAPure Compact Nucleic Acid Isolation Kit I or RNEASY® Mini Kit (QIAGEN®);NUCLISENS® NASBA Diagnostics (bioMérieux); or the MASTERPURE™ CompleteDNA and RNA Purification Kit (EPICENTRE)).

An array may be used to rapidly detect Demodex nucleic acids in asample. Arrays are arrangements of addressable locations on a substrate,with each address containing a nucleic acid, such as a probe, such as aDemodex probe. In some embodiments, each address corresponds to a singletype or class of nucleic acid, such as a single probe, though aparticular nucleic acid may be redundantly contained at multipleaddresses. A “microarray” is a miniaturized array requiring microscopicexamination for detection of hybridization. Larger “macroarrays” alloweach address to be recognizable by the naked human eye and, in someembodiments, a hybridization signal is detectable without additionalmagnification. The addresses may be labeled, keyed to a separate guide,or otherwise identified by location.

The use of the term “array” includes the arrays found in DNA microchiptechnology. As one, non-limiting example, the probes could be containedon a DNA microchip similar to the GENECHIP® products and relatedproducts commercially available from Affymetrix, Inc. (Santa Clara,Calif.). Briefly, a DNA microchip is a miniaturized, high-density arrayof probes on a glass wafer substrate. Particular probes are selected,and photolithographic masks are designed for use in a process based onsolid-phase chemical synthesis and photolithographic fabricationtechniques similar to those used in the semiconductor industry. Themasks are used to isolate chip exposure sites, and probes are chemicallysynthesized at these sites, with each probe in an identified locationwithin the array. After fabrication, the array is ready forhybridization. The probe or the nucleic acid within the sample may belabeled, such as with a fluorescent label and, after hybridization, thehybridization signals may be detected and analyzed.

Combination Treatments

Additional agents can be administered to the subject simultaneously orconsecutively with the spinosyn composition. Additional agents can beadministered before, during, or after topical administration of thespinosyn composition. The additional agents may be administered withinthe same composition as the one or more spinosyn compounds, oradministered to the subject in a separate composition. If administeredin a separate composition, the additional agents may be administeredtopically or by any local or systemic route appropriate for theadditional agents to have the desired effect on the subject. In someembodiments, the additional agent is administered topically. In otherembodiments, the additional agent is administered orally.

In some embodiments, the additional agent is one having miticidalactivity against the Demodex mite, such as one or more agents selectedfrom among ivermectin, pyrethrin, pyrethroid (e.g., permethrin,resmethrin, or D-phenothrin), tea tree oil (TTO), TTO component (e.g.,terpinen-4-ol (T4O)), metronidazole, hypochlorous acid (HOCl), essentialoil (e.g., peppermint oil or Salvia), alkali metal salt (e.g., lithiumsalt), phosphorothioate (e.g., a non-volatile, fat solublephosphorothioate such as coumaphos), or formamidine (e.g., amitraz, alsoidentified as N,N′-[(Methylimino)dimethylidyne]di-2,4-xylidine).

Permethrin is a member of the pyrethroids, which are a class ofsynthetically derived insecticides. Pyrethroids are structurally relatedto naturally occurring pyrethrins, pyrethrin I and pyrethrin II.Synthetic pyrethroids include permethrin (U.S. Pat. No. 4,113,968; U.S.Patent Publication 20160361297), resmethrin, and sumithrin (U.S. Pat.Nos. 3,934,023 and 2,348,930), which are each incorporated by referenceherein in their entirety. Other examples of pyrethroids includebifenthrin, cyfluthrin, cypermethrin, cyphenothrin, D-phenethrin,deltamethrin, esfenvalerate, etofenprox, fenprropathrin, flumethrin,gamma-cyhalothrin, imiprothrin, lambda-cyalothrin, momfluorothrin,permethrin, prallethrin, pyrethrin, tau-fluvalinate, tefluthrin, andtetramethrin.

In some embodiments, the additional agent is an antibiotic agent. Insome embodiments, the antibiotic agent has antibacterial activityagainst one or more bacteria found on or within the Demodex mite, suchas Streptococci, Staphylococci, or Bacillus oleronius. In someembodiments, the antibiotic agent also has miticidal activity, such asmetronidazole.

In some embodiments, the additional agent is one or moreanti-inflammatory agents. Examples of anti-inflammatory agents that maybe included are glucocorticoid or other steroid (e.g., prednisone,cortisone acetate, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone acetate,deoxycorticosterone acetate, aldosterone), non-steroidalanti-inflammatory drug (e.g., salicylates, arylalkanoic acids,2-arylpropionic acids, N-arylanthranilic acids, oxicams, coxibs, orsulphonanilides), Cox-2-specific inhibitor (e.g., valdecoxib, celecoxib,or rofecoxib), leflunomide, gold thioglucose, gold thiomalate, aurofin,sulfasalazine, hydroxychloroquinine, minocycline, TNF-alpha bindingprotein (e.g., infliximab, etanercept, or adalimumab), abatacept,anakinra, interferon-beta, interferon-gamma, interleukin-2, allergyvaccine, antihistamine, antileukotriene, beta-agonists, theophylline, oranticholinergic, antibiotics, tacrolimus, or retinoid.

In some embodiments, the anti-inflammatory agent is formulated fortopical use. In some embodiments, the topical anti-inflammatory agent isa topical steroid. In the U.S., topical steroids are classified(Class/Group I-VII) by their ability to constrict capillaries and causeskin blanching, with Group I being the strongest or most potent, andGroup VII being the weakest and mildest. Some examples of topicallyformulated steroids that may be utilized include clobetasol,betamethasone, diflorasone, fluocinonide, flurandrenolide, halobetasole,amcinonide, desoximetasone, halcinonide, fluticasone, triamcinolone,fluocinolone, hydrocortisone, mometasone, triamcinolone, alclometasone,denoside, and prednicarbate.

In some embodiments, the anti-inflammatory agent is formulated forophthalmic use. Some examples of ophthalmically formulated steroids thatmay be utilized include dexamethasone, difluprednate, fluoromethalone,loteprednol etabonate, and rimexolone.

In some embodiments, the addition& agent is one or more alkali metalsalts, which may be used as a miticidal agent. Lithium salts are thepreferred alkali metal salts. Lithium chloride is the most preferredalkali metal salt.

It should be understood that whenever a reference is made to alkalimetal salts or lithium salts, such alkali metal salts or lithium saltscomprise any organic as well as inorganic salts of alkali metals orlithium Which are suitable for and capable of exerting a miticidaleffect, preferably on the Demodex mite. Organic salts of alkali metalssuch as lithium include salts such as the citrate salt, carbonate salt,lactate salt, formate salt, acetate salt, trifluoroacetate salt, maleatesalt, tartrate salt, orotate salt and the like. Inorganic acid salts ofalkali metals include salts such as the fluoride salt, chloride salt,bromide salt, sulfate salt, phosphate salts and the like. For thepurposes of the present description, inorganic salts can be preferredover organic salts. Within the group of inorganic salts, the halides canbe preferred over other inorganic salts. Within the halides, thechloride salt can be particularly preferred. Irrespective of which saltsare used (organic salts versus inorganic salts, etc.), water-solublesalts are preferred as they can be provided in the form of abee-indigestible composition such as sucrose solutions which can be fedto the bees and thus taken up indirectly by the Varroa mites throughthis route. Whenever reference is made to a specific salt, it alwaysincludes the hydrated or anhydrous versions thereof unless indicatedotherwise. The group of alkali metals comprises lithium, sodium,potassium, rubidium and caesium. Within the group of alkali metals,lithium is particularly preferred.

Among the alkali metal salts, organic or inorganic salts of lithiumwhich may be hydrated or anhydrous are preferred over the correspondingsalts of e.g. potassium. Inorganic lithium salts such as LiCl areadvantageous because they cost less as compared to organic lithiumsalts. Within the group of lithium salts, inorganic lithium salts canthus be preferred over organic lithium salts. Preferred inorganiclithium salts include, but are not limited to, lithium chloride, lithiumbromide, lithium nitrate, lithium sulfate, lithium phosphate and thelike. However, organic lithium salts can be advantageous because theymay complex more than one lithium ion and because they may be taken upby the bees better as compared to inorganic lithium salts. Preferredorganic lithium salts include but are not limited to lithium citrate,lithium carbonate, lithium lactate, lithium formate, lithium acetate,lithium trifluoroacetate, lithium maleate, lithium tartrate, lithiumorotate and the like. Within the group of inorganic lithium salts, thehalides can be preferred over other inorganic salts. Within the group oforganic lithium salts, the lactates, citrates, acetates and carbonatescan be preferred over other organic salts. One alkali metal salt whichis preferred throughout all aspects and embodiments describedhereinafter is lithium chloride in hydrated or anhydrous form.

As with other additional agents, alkali metal salts may be administeredby a variety of methods. Preferably, the alkali metal salt is topicallyadministered to the ocular surface (conjunctiva and/or cornea) or otheranatomical structure of the eye, or to an area adjacent the eye. Thealkali metal salt may be included within the spinosyn composition oradministered to the subject in a separate composition before, during, orafter administration of the spinosyn composition. Preferably, amiticidally effective amount is administered. In some embodiment, theconcentration is within the range of about 0.5 mM to about 150 mM alkalimetal salt (e.g., LiCl).

Articles of Manufacture

Other aspects of the invention include articles of manufacture usefulfor carrying out the methods of the invention, such as the spinosyncomposition in association with a container; an ocular or facialapplicator; and a kit.

One aspect of the invention is an article of manufacture comprising aspinosyn composition disclosed herein; and a container with the topicalcomposition contained therein. Thus, the spinosyn compositions may becontained within a container, which may be accessed for administrationto a subject. The container may have a defined interior for containingthe composition. Preferably, the composition and container interior aresterile. Examples of containers include a collapsible or non-collapsibletube, bag, packet, blister, strip, ampoule, vial, bottle, can, or jar.The container may be composed of one or more materials appropriate forits use, such as aluminum, polyethylene (e.g., low-density polyethylene(LDPE), high-density polyethylene (HDPE), or polyethylene terephthalate(PET)), polypropylene, or glass.

In some embodiments, the container has a closure such as a cap, whichmay be actuated to access the composition within the container. Closuresused for the purpose of covering the container after the filling processshould be as inert as possible, not giving rise to undesiredinteractions between the contents and the outside environment, andshould provide a complete seal. In addition to their protectivefunction, closures should allow the easy and safe administration of thespinosyn composition. In some embodiments, the container has atamper-evident closure. In some embodiments, the container has noclosure and the spinosyn composition is accessed by tearing, separating,or breaking material of the container.

In some embodiments, the container includes a rubber closure (composedof elastomeric material), or a cap and/or overseal which may be composedof aluminum or plastic such as polyethylene, polypropylene.

The container may include a tamper-evident feature, such as atamper-evident closure. For example, an ophthalmic ointment is typicallysupplied in small, sterilized, collapsible tubes (containing, e.g., upto about 5 grams of the composition) fitted with a tamper-evidentapplicator. The tube may include a nozzle shaped so that the ointmentcan be administered without contaminating what remains in the tube. Thecontainer may include a reclosable child-resistant type of closure, suchas a “press-turn” or “squeeze-turn” type.

The container may be a single-dose or multi-dose container. In someembodiments, the container is a single-dose container (containing asingle dose of the composition for treating an ocular Demodex miteinfestation of an eye of the human or animal subject, or for treating acondition of the eye or skin associated with ocular Demodex miteinfestation). In other embodiments, the container is a multi-dosecontainer (containing multiple doses of the composition for treating anocular Demodex mite infestation of an eye of the human or animalsubject, or for treating a condition of the eye or skin associated withocular Demodex mite infestation).

Optionally, containers containing a composition described herein arelight-proof and have a tight seal. For example, the container(s) caninclude one of the dermatologically or ophthalmically acceptablespinosyn compositions described herein. In some embodiments, thecontainer protects against certain wavelengths of light and prolongedhigh temperature, and/or the ingress of air. In some embodiments, thecontainer is a sealed, light-proof container.

A label can be on or associated with the container. A label can be on acontainer when letters, numbers or other characters forming the labelare attached, molded or etched into the container itself; a label can beassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Alabel can be used to indicate that the contents are to be used for aspecific therapeutic application. The label can also indicate directionsfor use of the contents, such as in the methods described herein.

Another aspect of the invention is an ocular or facial applicator fortreating an ocular Demodex mite infestation of an eye of a human oranimal subject, or for treating a condition of the eye or skinassociated with ocular Demodex mite infestation. The applicator ispre-treated with, or contains, a spinosyn composition disclosed herein.The applicator may be used by the subject or by another individual totopically administer a composition comprising one or more spinosyncompounds to the ocular surface (conjunctiva and/or cornea) or otheranatomical structure of the eye, or to an area adjacent the eye. In someembodiments, the applicator is a swab, cosmetic pad, wipe, wipe stick,towelette, sponge, gauze, puff, wand, brush, or comb.

Another aspect of the invention is a kit for treating an ocular Demodexmite infestation of an eye of a human or animal subject, or for treatinga condition of the eye or skin associated with ocular Demodex miteinfestation. The kit comprises the spinosyn composition and an ocular orfacial applicator. The kit may further include a container disclosedherein containing the composition. Examples of applicators include aswab, cosmetic pad, wipe, wipe stick, towelette, sponge, gauze, puff,wand, brush, or comb.

In some embodiments, the applicator is pretreated with, or contains thecomposition. In this way, when the applicator is pretreated with, orcontains the composition, the applicator may function as a container aswell.

The kit may include a set of printed or digital instructions fortreating an ocular Demodex mite infestation of an eye of a human oranimal subject, or for treating a condition of the eye or skinassociated with ocular Demodex mite infestation, by topicallyadministering the composition to the ocular surface (conjunctiva and/orcornea) or other anatomical structure of the eye, or to an area adjacentthe eye. For example, the kit may include instructions for usecomprising the steps of topically applying the spinosyn composition toan affected area or other target area, and repeating the administrationstep until sufficient to alleviate or eliminate one or more signs orsymptoms associated with Demodex infestation, to reduce living Demodexcount, to eliminate living Demodex in a desired area, to reduce Demodexre-infestation, or to prevent or delay onset of Demodex infestation.

Kits can include packaging material that is compartmentalized to receiveone or more containers such as vials, tubes, and the like, each of thecontainer(s) including one of the separate elements to be used in amethod described herein. Packaging materials for use in packagingpharmaceutical products include, by way of example only U.S. Pat. Nos.5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packagingmaterials include, but are not limited to, blister packs, bottles,tubes, pumps, bags, vials, light-tight sealed containers, syringes,bottles, and any packaging material suitable for a selected formulationand intended mode of administration and treatment.

A kit may include one or more additional containers, each with one ormore of various materials desirable from a commercial and userstandpoint for use of the compositions for treating Demodex describedherein. Non-limiting examples of such materials include, but not limitedto, buffers, diluents, carrier, package, container, vial and/or tubelabels listing contents and/or instructions for use, and package insertswith instructions for use.

A label can be on or associated with the container. A label can be on acontainer when letters, numbers or other characters forming the labelare attached, molded or etched into the container itself; a label can beassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Alabel can be used to indicate that the contents are to be used for aspecific therapeutic application. The label can also indicate directionsfor use of the contents, such as in the methods described herein.

In some embodiments of the kit, the spinosyn composition can bepresented in a pack or dispenser device which can contain one or moreunit dosage forms containing a spinosyn composition disclosed herein.The pack can for example contain metal or plastic foil, such as ablister pack. The pack or dispenser device can be accompanied byinstructions for administration. The pack or dispenser can also beaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.Such notice, for example, can be the labeling approved by the U.S. Foodand Drug Administration for prescription drugs, or the approved productinsert. Compositions containing a spinosyn composition provided hereinformulated in a compatible pharmaceutical carrier can also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition.

Definitions

As used herein, the term “amplify” refers to increasing the number ofcopies of a target nucleic acid molecule. The resulting amplificationproducts are called “amplicons.” Amplification of a nucleic acidmolecule (such as a DNA or RNA molecule) refers to use of a techniquethat increases the number of copies of a nucleic acid molecule in asample, for example the number of copies of a Demodex (e.g., D.folliculorum or D. brevis) nucleic acid. An example of amplification isthe polymerase chain reaction (PCR), in which a sample is contacted witha pair of oligonucleotide primers under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, re-annealed, extended, and dissociated to amplify the numberof copies of the nucleic acid. This cycle can be repeated. The productof amplification can be characterized by such techniques aselectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing.

Primer pairs can be used for amplification of a target nucleic acidsequence, for example, by PCR, real-time PCR, or other nucleic-acidamplification methods known in the art. An “upstream” or “forward”primer is a primer 5′ to a reference point on a nucleic acid sequence. A“downstream” or “reverse” primer is a primer 3′ to a reference point ona nucleic acid sequence. In general, at least one forward and onereverse primer are included in an amplification reaction. PCR primerpairs can be derived from a known sequence (e.g., a Demodex sequence),for example, by using computer programs intended for that purpose suchas Primer (Version 0.5, © 1991, Whitehead Institute for BiomedicalResearch, Cambridge, Mass.) or PRIMER EXPRESS® Software (AppliedBiosystems, AB, Foster City, Calif.).

Methods for preparing and using primers are described in, for example,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y.; Ausubel et al. (1987) Current Protocols inMolecular Biology, Greene Publ. Assoc. & Wiley-Intersciences. In oneexample, a primer includes a label.

Other examples of in vitro amplification techniques include quantitativereal-time PCR; reverse transcriptase PCR (RT-PCR); real-time PCR;real-time reverse transcriptase PCR (rt RT-PCR); nested PCR; stranddisplacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881, repair chain reaction amplification (see WO 90/01069); ligasechain reaction amplification (see European patent publication EP-A-320308); gap filling ligase chain reaction amplification (see U.S. Pat. No.5,427,930); coupled ligase detection and PCR (see U.S. Pat. No.6,027,889); and NASBA™ RNA transcription free amplification (see U.S.Pat. No. 6,025,134), among others.

As used herein, the term “detect” refers to the determination of whetheran agent (such as a signal, particular nucleotide, amino acid, nucleicacid molecule, and/or organism) is present or absent, for example, aDemodex mite such as D. folliculorum and/or D. brevis. In some examples,this can further include quantification. For example, use of probespermits detection of a fluorophore, for example, detection of a signalfrom a fluorophore, which can be used to determine if a nucleic acidcorresponding to nucleic acid of Demodex (D. folliculorum and/or D.brevis) is present. The detection of a D. folliculorum and/or D. brevisnucleic acid molecule indicates the presence of D. folliculorum and/orD. brevis in the sample, for example a D. folliculorum and/or D. brevisinfestation.

The terms “acaricidal” and “miticidal” are used herein interchangeablyto refer to the ability to kill mites of the Demodex genus in any lifestage, or the ability to interfere with a Demodex mite's growth or lifecycle in any way that results in an overall reduction in Demodex mitepopulation. In some embodiments, the Demodex species is D. folliculorum,D. brevis, or both. For example, the term “miticidal” includesinhibition or elimination of reproductive ability of a pest, as well asinhibition of a pest from progressing from one form to a more matureform, e.g., transition between various larval forms or transition fromlarvae to proto-nymph or from proto-nymph to nymph, or from nymph toadult. Further, the term miticidal is intended to include all phases ofa mite life cycle; thus, for example, the term includes larvicidal,ovicidal, and adulticidal action.

As used herein, the term “miticidally effective” is intended to indicatean amount or concentration of a spinosyn or other miticide that issufficient to reduce the number of Demodex mites in an anatomical locus,as compared to a corresponding anatomical locus in the absence of theamount or concentration of the spinosyn or other miticide.

As used herein, the term “treatment”, “treating”, and grammaticalvariations thereof refer to an amelioration, prophylaxis, or reversal ofa disease or disorder, or of at least one sign or symptom thereof. Insome embodiments, “treatment” or “treating” refers to an amelioration,prophylaxis, or reversal of at least one measurable physical parameterrelated to the disease or disorder being treated, not necessarilydiscernible in or by the subject. In yet another embodiment, “treatment”or “treating” refers to inhibiting or slowing the progression of adisease or disorder, either physically, e.g., stabilization of adiscernible sign or symptom, physiologically, e.g., stabilization of aphysical parameter, or both. In yet another embodiment, “treatment” or“treating” refers to delaying the onset of a disease or disorder.

EXEMPLIFIED EMBODIMENTS

1. A method for treating an ocular Demodex mite infestation of an eye ofa human or animal subject, or for treating a condition of the eye orskin associated with ocular Demodex mite infestation, comprisingtopically administering a composition comprising one or more spinosyncompounds to the ocular surface (conjunctiva and/or cornea) or otheranatomical structure of the eye, or to an area adjacent the eye.

2. The method of embodiment 1, wherein the subject has the condition,and the condition is one or more from among Demodex-induced blepharitis(also called Demodex blepharitis), Demodex-induced ocular rosacea,Demodex-induced facial rosacea, dry eye, meibomian gland dysfunction,chalazion, hordeolum, follicular inflammation, non-specific facialdermatitis, infiltrative keratoconjunctivitis, nodular scar deposition,or corneal neovascularization.

3. The method of embodiment 1 or 2, wherein the treatment alleviates oneor more signs or symptoms in the subject selected from among: itching,burning, foreign body sensation, crusting and redness of the lid margin,blurry vision, cylindrical dandruff, eyelash misalignment, eyelashtrichiasis, eyelash madarosis, lid margin inflammation, meibomian glanddysfunction, blepharoconjunctivitis, and blepharokeratitis.

4. The method of any one of embodiments 1 to 3, wherein the compositionis topically administered to an area adjacent to the eye, wherein thearea comprises one or more of an eyelid, eyelid margin, eyelashes,eyelash follicles, eyebrow, or eyebrow follicles.

5. The method of any one of embodiments 1 to 3, wherein the compositionis topically administered to an area adjacent to the eye, wherein thearea comprises a sebaceous gland opening of the eyelid (e.g., one ormore of gland of Zeis, gland of Moll, or Meibomian gland).

6. The method of any preceding embodiment, wherein the Demodex mitecomprises Demodex folliculorum, Demodex brevis, or both.

7. The method of any preceding embodiment, wherein the subject issymptomatic at the time of said administering.

8. The method of any one of embodiments 1 to 6, wherein the subject isasymptomatic at the time of said administering.

9. The method of any preceding embodiment, further comprising confirmingthe presence of Demodex mites prior to said administering.

10. The method of embodiment 9, wherein the said confirming comprisesidentifying the Demodex mites in vivo or in a biological sample (e.g.,hair or skin sample) ex vivo, or detecting a molecule associated withthe presence of Demodex mites (e.g., chitin, chitin synthase, lipase,Bacillus antigen).

11. The method of any preceding embodiment, wherein an effective amountof the composition is topically administered to reduce the number ofliving Demodex mites on the eye and adjacent skin and hair.

12. The method of any preceding embodiment, wherein an effective amountof the composition is topically administered to eliminate the presenceof living Demodex mites from the eye and adjacent skin and hair.

13. The method of any preceding embodiment, wherein the one or morespinosyn compounds have the chemical structure of formula (I) or apharmaceutically acceptable salt thereof:

where R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵,and R¹⁶, can be independently selected from the group consisting of:null; H; F; Cl; Br; I; OH; CN; (C₁₋₄)alkyl, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;(C₂₋₄)alkenyl, such as ethenyl, propenyl, butenyl, where the double bondcan be located at any position in the alkenyl carbon chain, andincluding any alkenyl conformational isomers; alkynyl; aralkyl; alkaryl;halogenated alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl;cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl; amino; alkylamino;arylamino; dialkylamino; alkylarylamino; diarylamino; acylamino;hydroxyl; thiol; thioalkyl; alkoxy; alkylthio; alkoxyalkyl; aryloxy;arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy;benzyloxy; phosphonic acid; phosphate ester; sulfonic acid (—SO₃H);sulfonate ester; sulfonamide; alkaryl; arylalkyl; carbamate; amino;alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino;alkylthio; heteroalkyl; alkyltriphenylphosphonium; heterocyclyl; ketone(═O); ether (—OR¹⁷); and ester (—COOR¹⁸ and —OC(═O)R¹⁸);

where R⁵ and R⁷ can be a double bond within the cyclopentane ring;

where R¹¹ and R¹³ can be a double bond within the cyclohexane ring;

where R¹⁷ can be independently selected from the group consisting of: a(C₁₋₄)alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl; (C₂₋₄)alkenyl, such as ethenyl,propenyl, butenyl, where the double bond can be located at any positionin the alkenyl carbon chain, and including any alkenyl conformationalisomers; and alkynyl;

where R¹⁸ can be independently selected from the group consisting of: a(C₁₋₄)alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl; (C₂₋₄)alkenyl, such as ethenyl,propenyl, butenyl, where the double bond can be located at any positionin the alkenyl carbon chain, and including any alkenyl conformationalisomers; and alkynyl.

14. The method of any preceding embodiment, wherein the one or morespinosyn compounds are selected from among spinosyn A, spinosyn B,spinosyn C, spinosyn D, spinosyn E, spinosyn F, spinosyn G, spinosyn H,spinosyn I, spinosyn J, spinosyn K, spinosyn L, spinosyn M, spinosyn N,spinosyn O, spinosyn P, spinosyn R, spinosyn S, spinosyn T, spinosyn U,spinosyn V, spinosyn W, spinosyn Y, spinosoid N-demethyl D, spinosoidN-demethyl K, spinosoid N,N-didimethyl K, spinosoid N-demethyl P,spinosoid 2′-H A, spinosoid 2′-H D, spinosoid 2′-O-ethyl A, spinosoid3′-H A, spinosoid 3′-O-ethyl A, spinosoid 3′-O-n-propyl A, spinosoid3′-O-n-butyl A, spinosoid 3′-O-allyl A, spinosoid —O—CH₂CF₃A, spinosoid4′-H A, spinosoid 4′-O-ethyl A, or spinosoid 2′,3′,4′-tri-O-ethyl A.

15. The method of any preceding embodiment, wherein the one or morespinosyn compounds comprise a combination of spinosyn A and spinosyn D.

16. The method of embodiment 15, wherein the spinosyn A and spinosyn Dare present in the composition in a ratio of about 18:2 to about 16:4spinosyn A to spinosyn D.

17. The method of embodiment 15, wherein the spinosyn A and spinosyn Dare present in the composition in a ratio of about 17:3 spinosyn A tospinosyn D.

18. The method of any preceding embodiment, wherein the compositionincludes no active agent other than the one or more spinosyn compounds.

19. The method of any one of embodiment 1 to 18, wherein the compositioncomprises no additional agent that has miticidal activity against theDemodex mite.

20. The method of any one of embodiments 1 to 17, wherein the methoddoes not include topical administration of any active agent other thanthe one or more spinosyn compounds to the ocular surface (conjunctivaand/or cornea) or other anatomical structure of the eye, or to an areaadjacent the eye.

21. The method of any one of embodiments 1 to 17, wherein the methoddoes not include topical administration of an agent having miticidalactivity against the Demodex mite in addition to the one or morespinosyn compounds to the ocular surface (conjunctiva and/or cornea) orother anatomical structure of the eye, or to an area adjacent the eye.

22. The method of any one of embodiments 1 to 17, wherein thecomposition further comprises an additional agent having miticidalactivity against the Demodex mite.

23. The method of embodiment 22, wherein the additional agent havingmiticidal activity against the Demodex mite is one or more agentsselected from among ivermectin, pyrethrin, pyrethroid (e.g., permethrin,resmethrin, or D-phenothrin), tea tree oil (TTO), TTO component (e.g.,terpinen-4-ol (T4O)), metronidazole, hypochlorous acid (HOCl), essentialoil (e.g., peppermint oil or Salvia), alkali metal salt (e.g., lithiumsalt), phosphorothioate (e.g., a non-volatile, fat solublephosphorothioate such as coumaphos), or formamidine (e.g., amitraz).

24. The method of any one of embodiments 1 to 17, further comprisingadministering an additional agent having miticidal activity against theDemodex mite before, during, or after topical administration of thecomposition with the one or more spinosyn compounds, wherein theadditional agent is administered in a separate composition.

25. The method of embodiment 24, wherein the additional agent havingmiticidal activity against the Demodex mite is one or more agentsselected from among ivermectin, pyrethrin, pyrethroid (e.g., permethrin,resmethrin, or D-phenothrin), tea tree oil (TTO), TTO component (e.g.,terpinen-4-ol (T4O)), metronidazole, hypochlorous acid (HOCl), essentialoil (e.g., peppermint oil or Salvia), alkali metal salt (e.g., lithiumsalt), phosphorothioate (e.g., a non-volatile, fat solublephosphorothioate such as coumaphos), or formamidine (e.g., amitraz).

26. The method of any preceding embodiment, further comprisingadministering an antibiotic agent to the subject, wherein the antibioticagent is in the composition with the one or more spinosyn compounds orin a separate composition.

27. The method of embodiment 26, wherein the antibiotic agent hasantibacterial activity against one or more of Streptococci,Staphylococci, or Bacillus oleronius.

28. The method of any preceding embodiment, further comprisingadministering an anti-inflammatory agent to the subject, wherein theanti-inflammatory agent is in the composition with the one or morespinosyn compounds or in a separate composition.

29. The method of embodiment 28, wherein the anti-inflammatory agent isselected from among a glucocorticoid or other steroid (e.g., prednisone,cortisone acetate, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone acetate,deoxycorticosterone acetate, aldosterone), non-steroidalanti-inflammatory drug (e.g., salicylates, arylalkanoic acids,2-arylpropionic acids, N-arylanthranilic acids, oxicams, coxibs, orsulphonanilides), Cox-2-specific inhibitor (e.g., valdecoxib, celecoxib,or rofecoxib), leflunomide, gold thioglucose, gold thiomalate, aurofin,sulfasalazine, hydroxychloroquinine, minocycline, TNF-alpha bindingprotein (e.g., infliximab, etanercept, or adalimumab), abatacept,anakinra, interferon-beta, interferon-gamma, interleukin-2, allergyvaccine, antihistamine, antileukotriene, beta-agonists, theophylline, oranticholinergic, antibiotics, tacrolimus, or retinoid.

30. The method of any preceding embodiment, wherein the composition is asolution, suspension, salve, spray, lotion, gel, paste, balm, foam,mousse, scrub or cleanser (e.g., shampoo or soap), cream, or ointment.

31. The method of any preceding embodiment, wherein the composition istopically administered using an applicator (e.g., swab, cosmetic pad,wipe, wipe stick, towelette, sponge, gauze, puff, wand, brush, or comb).

32. The method of any preceding embodiment, wherein the composition is acosmetic product.

33. The method of embodiment 32, wherein the cosmetic product is amascara, eye shadow, or eye liner.

34. The method of any preceding embodiment, wherein the compositionfurther comprises one or more excipients selected from among petrolatum,mineral oil, propylparaben, methylparaben, lanolin, chlorobutanol,water, lanolin alcohol, sodium thiosulfate, sodium phosphate monobasic,phenylmercuric acetate, mannitol, zinc chloride, sodium phosphate,potassium acetate, hypromelloses, gentamcicin sulfate, boric acid,sodium hydroxide, lanolin oil, carbomer homopolymer type A or B (allylpentaerythritol crosslinked), or benzalkonium chloride.

35. The method of any one of embodiments 1 to 34, wherein thecomposition is an emulsion and further comprises one or more excipientsselected from among water, sodium hydroxide, polysorbate 80, glycerine,castor oil, carbomer copolymer type A, sodium acetate, boric acid,sorbic acid, edetate disodium, or silicone oil or silicone polymer gel(e.g., dimethicone or cyclomethicone).

36. The method of any preceding embodiment, wherein the compositionfurther comprises a gelling agent (e.g., gum, agar, carrageenan,petrolatum, or a cellulosic polymer (e.g., hydroxyethyl cellulose orhydroxymethyl cellulose)).

37. The method of any preceding embodiment, further comprising one ormore of a solvent, co-solvent, demulcent, emollient, preservative,antioxidant, moisturizer, or solubilizing agent.

38. The method of any preceding embodiment, wherein compositioncomprises 0.1% to 10% (w/v) of the one or more spinosyn compounds.

39. The method of any preceding embodiment, wherein the composition isaccessed and dispensed from a container, with or without a closure(e.g., a cap), prior to administration.

40. The method of embodiment 39, wherein the container is a collapsibleor non-collapsible tube, bag, packet, blister, strip, ampoule, viral,bottle, can, or jar.

41. The method of embodiment 39 or 40, wherein the container comprisesone or more materials selected from among aluminum, polyethylene (e.g.,low-density polyethylene (LDPE), high-density polyethylene (HDPE), orpolyethylene terephthalate (PET)), polypropylene, or glass.

42. The method of any one of embodiments 39 to 41, wherein the containeris a single-dose container (containing a single dose of the compositionfor treating an ocular Demodex mite infestation of an eye of the humanor animal subject, or for treating a condition of the eye or skin causedassociated with ocular Demodex mite infestation).

43. The method of any one of embodiments 39 to 41, wherein the containeris a multi-dose container (containing multiple doses of the compositionfor treating an ocular Demodex mite infestation of an eye of the humanor animal subject, or for treating a condition of the eye or skin causedassociated with ocular Demodex mite infestation).

44. The method of any one of embodiments 39 to 43, wherein the containerhas an interior containing the composition, and wherein the containerinterior and the composition are sterile.

45. The method of any preceding embodiment, wherein the composition is acomposition of one of Table 1, Table 2, Table 3, Table 4, or Table 5.

46. A topical composition comprising 0.1% to 10% (w/v) of the one ormore spinosyn compounds.

47. The topical composition of embodiment 46, wherein the one or morespinosyn compounds have the chemical structure of formula (I) or apharmaceutically acceptable salt thereof:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, andR¹⁶, can be independently selected from the group consisting of: null;H; F; Cl; Br; I; OH; CN; (C₁₋₄)alkyl, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C₂₋₄)alkenyl, suchas ethenyl, propenyl, butenyl, where the double bond can be located atany position in the alkenyl carbon chain, and including any alkenylconformational isomers; alkynyl; aralkyl; alkaryl; halogenated alkyl;heteroalkyl; aryl; heterocyclyl; cycloalkyl; cycloalkenyl; cycloalkynyl;hydroxyalkyl; aminoalkyl; amino; alkylamino; arylamino; dialkylamino;alkylarylamino; diarylamino; acylamino; hydroxyl; thiol; thioalkyl;alkoxy; alkylthio; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro;carbamoyl; trifluoromethyl; phenoxy; benzyloxy; phosphonic acid;phosphate ester; sulfonic acid (—SO₃H); sulfonate ester; sulfonamide;alkaryl; arylalkyl; carbamate; amino; alkylamino; arylamino;dialkylamino; alkylarylamino; diarylamino; alkylthio; heteroalkyl;alkyltriphenylphosphonium; heterocyclyl; ketone (═O); ether (—OR¹⁷); andester (—COOR¹⁸ and —OC(═O)R¹⁸);

where R⁵ and R⁷ can be a double bond within the cyclopentane ring;

where R¹¹ and R¹³ can be a double bond within the cyclohexane ring;

where R¹⁷ can be independently selected from the group consisting of: a(C₁₋₄)alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl; (C₂₋₄)alkenyl, such as ethenyl,propenyl, butenyl, where the double bond can be located at any positionin the alkenyl carbon chain, and including any alkenyl conformationalisomers; and alkynyl;

where R¹⁸ can be independently selected from the group consisting of: a(C₁₋₄)alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl; (C₂₋₄)alkenyl, such as ethenyl,propenyl, butenyl, where the double bond can be located at any positionin the alkenyl carbon chain, and including any alkenyl conformationalisomers; and alkynyl.

48. The topical composition of embodiment 46 or 47, wherein the one ormore spinosyn compounds comprise a combination of spinosyn A andspinosyn D.

49. The topical composition of embodiment 48, wherein the spinosyn A andspinosyn D are present in the composition in a ratio of about 18:2 toabout 16:4 spinosyn A to spinosyn D.

50. The topical composition of embodiment 48, wherein the spinosyn A andspinosyn D are present in the composition in a ratio of about 17:3spinosyn A to spinosyn D.

51. The topical composition of any one of embodiments 46 to 50, whereinthe composition includes no active agent other than the one or morespinosyn compounds.

52. The topical composition of any one of embodiments 46 to 50, whereinthe composition comprises no additional agent that has miticidalactivity against the Demodex mite.

53. The topical composition of any one of embodiments 46 to 50, whereinthe composition further comprises an additional agent having miticidalactivity against the Demodex mite.

54. The topical composition of embodiment 53, wherein the additionalagent having miticidal activity against the Demodex mite is one or moreagents selected from among ivermectin, pyrethrin, pyrethroid (e.g.,permethrin, resmethrin, or D-phenothrin), tea tree oil (TTO), TTOcomponent (e.g., terpinen-4-ol (T4O)), metronidazole, hypochlorous acid(HOCl), essential oil (e.g., peppermint oil or Salvia), alkali metalsalt (e.g., lithium salt), phosphorothioate (e.g., a non-volatile, fatsoluble phosphorothioate such as coumaphos), or formamidine (e.g.,amitraz).

55. The topical composition of any one of embodiments 46 to 54, furthercomprising an antibiotic agent.

56. The topical composition of embodiment 55, wherein the antibioticagent has antibacterial activity against one or more of Streptococci,Staphylococci, or Bacillus oleronius.

57. The topical composition of any one of embodiments 46 to 56, furthercomprising an anti-inflammatory agent.

58. The topical composition of embodiment 57, wherein theanti-inflammatory agent is selected from among a glucocorticoid or othersteroid (e.g., prednisone, cortisone acetate, prednisolone,methylprednisolone, dexamethasone, betamethasone, triamcinolone,beclometasone, fludrocortisone acetate, deoxycorticosterone acetate,aldosterone), non-steroidal anti-inflammatory drug (e.g., salicylates,arylalkanoic acids, 2-arylpropionic acids, N-arylanthranilic acids,oxicams, coxibs, or sulphonanilides), Cox-2-specific inhibitor (e.g.,valdecoxib, celecoxib, or rofecoxib), leflunomide, gold thioglucose,gold thiomalate, aurofin, sulfasalazine, hydroxychloroquinine,minocycline, TNF-alpha binding protein (e.g., infliximab, etanercept, oradalimumab), abatacept, anakinra, interferon-beta, interferon-gamma,interleukin-2, allergy vaccine, antihistamine, antileukotriene,beta-agonists, theophylline, or anticholinergic, antibiotics,tacrolimus, or retinoid.

59. The topical composition of any one of embodiments 46 to 58, whereinthe composition is a solution, suspension, salve, spray, lotion, gel,paste, balm, foam, mousse, scrub or cleanser (e.g., shampoo or soap),cream, or ointment.

60. The topical composition of any one of embodiments 46 to 59, whereinthe composition is a cosmetic product.

61. The topical composition of embodiment 60, wherein the composition isa mascara, eye shadow, or eye liner.

62. The topical composition of any one of embodiments 46 to 61, whereinthe composition is an ointment and further comprises one or moreexcipients selected from among petrolatum, mineral oil, propylparaben,methylparaben, lanolin, chlorobutanol, water, lanolin alcohol, sodiumthiosulfate, sodium phosphate monobasic, phenylmercuric acetate,mannitol, zinc chloride, sodium phosphate, potassium acetate,hypromelloses, gentamcicin sulfate, boric acid, sodium hydroxide,lanolin oil, carbomer homopolymer type b (allyl pentaerythritolcrosslinked), or benzalkonium chloride.

63. The topical composition of any one of embodiments 46 to 61, whereinthe composition is an emulsion and further comprises one or moreexcipients selected from among water, sodium hydroxide, polysorbate 80,glycerine, castor oil, carbomer copolymer type A, sodium acetate, boricacid, sorbic acid, edetate disodium, carbomer copolymer type a (allylpentaerythritol crosslinked), or silicone oil or silicone polymer gel(e.g., dimethicone or cyclomethicone).

64. The topical composition of any one of embodiments 46 to 63, whereinthe composition further comprises a gelling agent (e.g., gum, agar,carrageenan, petrolatum, or a cellulosic polymer (e.g., hydroxyethylcellulose or hydroxymethyl cellulose)).

65. The topical composition of any one of embodiments 46 to 64, furthercomprising one or more of a solvent, co-solvent, demulcent, emollient,preservative, antioxidant, moisturizer, or solubilizing agent.

66. The topical composition of any one of embodiments 46 to 64, whereinthe composition is a composition of one of Table 1, Table 2, Table 3,Table 4, or Table 5.

67. An article of manufacture comprising the topical composition of anyone of embodiments 46 to 66; and a container with the topicalcomposition contained therein.

68. The article of embodiment 67, wherein the container has a closure(e.g., a cap), which may be actuated to access the composition withinthe container.

69. The article of embodiment 67 or 68, wherein the container is acollapsible or non-collapsible tube, bag, packet, blister, strip,ampoule, vial, bottle, can, or jar.

70. The article of any one of embodiments 67 to 69, wherein thecontainer comprises one or more materials selected from among aluminum,polyethylene (e.g., low-density polyethylene (LDPE), high-densitypolyethylene (HDPE), or polyethylene terephthalate (PET)),polypropylene, or glass.

71. The article of any one of embodiments 67 to 70, wherein thecontainer is a single-dose container (containing a single dose of thecomposition for treating an ocular Demodex mite infestation of an eye ofthe human or animal subject, or for treating a condition of the eye orskin associated with ocular Demodex mite infestation).

72. The article of any one of embodiments 67 to 70, wherein thecontainer is a multi-dose container (containing multiple doses of thecomposition for treating an ocular Demodex mite infestation of an eye ofthe human or animal subject, or for treating a condition of the eye orskin associated with ocular Demodex mite infestation).

73. The article of any one of embodiments 67 to 72, wherein thecontainer has an interior containing the composition, and wherein thecontainer interior and the composition are sterile.

74. An ocular or facial applicator pre-treated with, or containing, thetopical composition of any one of embodiments 46 to 66.

75. The ocular or facial applicator of embodiment 74, wherein theapplicator is a swab, cosmetic pad, wipe, wipe stick, towelette, sponge,gauze, puff, wand, brush, or comb.

76. A kit comprising the topical composition of any one of embodiments46 to 66; and an ocular or facial applicator.

77. The kit of embodiment 76, wherein the kit further comprises acontainer containing the composition. 78. The kit of embodiment 76,wherein the applicator is pretreated with, or containing thecomposition.

79. The kit of any one of embodiments 76 to 78, wherein the applicatoris a swab, cosmetic pad, wipe, wipe stick, towelette, sponge, gauze,puff, wand, brush, or comb.

80. The kit of any one of embodiments 76 to 79, further comprisinginstructions for treating an ocular Demodex mite infestation of an eyeof a human or animal subject, or for treating a condition of the eye orskin associated with ocular Demodex mite infestation, by topicallyadministering the composition to the ocular surface (conjunctiva and/orcornea) or other anatomical structure of the eye, or to an area adjacentthe eye.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all FIGURES and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

What is claimed is:
 1. A method for treating a condition, sign, orsymptom of the eye or skin associated with ocular Demodex miteinfestation of a human or animal subject, the method comprisingtopically administering a miticidally effective amount of a compositioncomprising one or more spinosyn compounds to one or more anatomicalsites of the eye of the subject having the condition, sign, or symptom,wherein the one or more anatomical sites are selected from among aneyelid, eyelid margin, eyelash, eyelash follicles, eyebrow, eyebrowfollicles, or corner of the eye.
 2. The method of claim 1, wherein thesubject has the condition, and the condition is one or more from amongDemodex-induced blepharitis (also called Demodex blepharitis),Demodex-induced ocular rosacea, Demodex-induced facial rosacea, dry eye,meibomian gland dysfunction, chalazion, hordeolum, follicularinflammation, non-specific facial dermatitis, infiltrativekeratoconjunctivitis, nodular scar deposition, or cornealneovascularization.
 3. The method of claim 1, wherein the subject hasthe sign or symptom, and the sign or symptom is selected from among:itching of the eye, burning sensation of the eye, foreign body sensationof the eye, crusting and redness of the lid margin, blurry vision,cylindrical dandruff, eyelash misalignment, eyelash trichiasis, eyelashmadarosis, lid margin inflammation, meibomian gland dysfunction,blepharoconjunctivitis, and blepharokeratitis.
 4. The method of claim 1,wherein the composition comprises no additional agent that has activityagainst the Demodex mite.
 5. The method of claim 1, wherein thecomposition includes no active agent other than the one or more spinosyncompounds.
 6. The method of claim 2, wherein the condition isDemodex-induced blepharitis.
 7. The method of claim 2, wherein thecondition is Demodex-induced ocular rosacea.
 8. The method of claim 2,wherein the condition is Demodex-induced facial rosacea.
 9. The methodof claim 2, wherein the condition is dry eye.
 10. The method of claim 2,wherein the condition is a chalazion or hordeolum.
 11. The method ofclaim 1, wherein the subject has the sign or symptom, and the sign orsymptom is hyperemia or telangiectasia of the eye lid.
 12. The method ofclaim 3, wherein the sign or symptom is one or more selected from amongeyelash misalignment, eyelash trichiasis, or eyelash madarosis.
 13. Themethod of claim 3, wherein the sign or symptom is meibomian glanddysfunction.
 14. The method of claim 3, wherein the sign or symptom isitching of the eye, burning sensation of the eye, or foreign bodysensation of the eye.
 15. The method of claim 3, wherein the sign orsymptom is lid margin inflammation.
 16. The method of claim 3, whereinthe sign or symptom is blepharoconjunctivitis or blepharokeratitis. 17.The method of claim 1, wherein the one or more spinosyn compounds havethe chemical structure of formula (I) or a pharmaceutically acceptablesalt thereof:

where R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵,and R¹⁶, can be independently selected from the group consisting of:null; H; F; Cl; Br; I; OH; CN; (C₁₋₄)alkyl, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;(C₂₋₄)alkenyl, such as ethenyl, propenyl, butenyl, where the double bondcan be located at any position in the alkenyl carbon chain, andincluding any alkenyl conformational isomers; alkynyl; aralkyl; alkaryl;halogenated alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl;cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl; amino; alkylamino;arylamino; dialkylamino; alkylarylamino; diarylamino; acylamino;hydroxyl; thiol; thioalkyl; alkoxy; alkylthio; alkoxyalkyl; aryloxy;arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy;benzyloxy; phosphonic acid; phosphate ester; sulfonic acid (—SO₃H);sulfonate ester; sulfonamide; alkaryl; arylalkyl; carbamate; amino;alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino;alkylthio; heteroalkyl; alkyltriphenylphosphonium; heterocyclyl; ketone(═O); ether (—OR¹⁷); and ester (—COOR¹⁸ and —OC(═O)R¹⁸); where R⁵ and R⁷can be a double bond within the cyclopentane ring; where R¹¹ and R¹³ canbe a double bond within the cyclohexane ring; where R¹⁷ can beindependently selected from the group consisting of: a (C₁₋₄)alkyl, suchas methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,tert-butyl; (C₂₋₄)alkenyl, such as ethenyl, propenyl, butenyl, where thedouble bond can be located at any position in the alkenyl carbon chain,and including any alkenyl conformational isomers; and alkynyl; where R¹⁸can be independently selected from the group consisting of: a(C₁₋₄)alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl; (C₂₋₄)alkenyl, such as ethenyl,propenyl, butenyl, where the double bond can be located at any positionin the alkenyl carbon chain, and including any alkenyl conformationalisomers; and alkynyl.
 18. The method of claim 1, wherein the one or morespinosyn compounds comprise a combination of spinosyn A and spinosyn D.19. The method of claim 1, wherein the one or more spinosyn compounds isspinosad.
 20. The method of claim 1, wherein the Demodex mite comprisesDemodex folliculorum mites, Demodex brevis mites, or both Demodexfolliculorum mites and Demodex brevis mites.